Method of synthesizing nucleic acid

ABSTRACT

The present invention relates to an oligonucleotide having a novel structure and a method of synthesizing nucleic acid by using the same as a primer. This oligonucleotide is provided at the 5′-side of the primer with a nucleotide sequence substantially the same as a region synthesized with this primer as the origin of synthesis. The present invention realizes synthesis of nucleic acid based on an isothermal reaction with a simple constitution of reagents. Further, the present invention provides a method of synthesizing highly specific nucleic acid on the basis of this method of synthesizing nucleic acid.

TECHNICAL FIELD

[0001] The present invention relates to a method of synthesizing nucleicacid composed of a specific nucleotide sequence, which is useful as amethod of amplifying nucleic acid.

BACKGROUND ART

[0002] An analysis method based on complementarity of a nucleic acidnucleotide sequence can analyze genetic traits directly. Accordingly,this analysis is a very powerful means for identification of geneticdiseases, canceration, microorganisms etc. Further, a gene itself is theobject of detection, and thus time-consuming and cumbersome proceduressuch as in culture can be omitted in some cases.

[0003] Nevertheless, the detection of a target gene present in a verysmall amount in a sample is not easy in general so that amplification ofa target gene itself or its detection signal is necessary. As a methodof amplifying a target gene, the PCR (polymerase chain reaction) methodis known. (Science, 230, 1350-1354, 1985). Currently, the PCR method isthe most popular method as a technique of amplifying nucleic acid invitro. This method was established firmly as an excellent detectionmethod by virtue of high sensitivity based on the effect of exponentialamplification. Further, since the amplification product can be recoveredas DNA, this method is applied widely as an important tool supportinggenetic engineering techniques such as gene cloning and structuraldetermination. In the PCR method, however, there are the following notedproblems: a special temperature controller is necessary for practice;the exponential progress of the amplification reaction causes a problemin quantification; and samples and reaction solutions are easilycontaminated from the outside to permit nucleic acid mixed in error tofunction as a template.

[0004] As genomic information is accumulated, analysis of singlenucleotide polymorphism (SNPs) comes to attract attention. Detection ofSNPs by means of PCR is feasible by designing a primer such that itsnucleotide sequence contains SNPs. That is, whether a nucleotidesequence complementary to the primer is present or not can be inferredby determining whether a reaction product is present or not. However,once a complementary chain is synthesized in error in PCR by any chance,this product functions as a template in subsequent reaction, thuscausing an erroneous result. In practice, it is said that strict controlof PCR is difficult with the difference of only one base given at theterminal of the primer. Accordingly, it is necessary to improvespecificity in order to apply PCR to detection of SNPs.

[0005] On one hand, a method of synthesizing nucleic acid by a ligase isalso practically used. The LCR method (ligase chain reaction, Laffler TG; Garrino J J; Marshall R L; Ann. Biol. Clin. (Paris), 51:9, 821-6,1993) is based on the reaction in which two adjacent probes arehybridized with a target sequence and ligated to each other by a ligase.The two probes could not be ligated in the absence of the targetnucleotide sequence, and thus the presence of the ligated product isindicative of the target nucleotide sequence. Because the LCR methodalso requires control of temperature for separation of a complementarychain from a template, there arises the same problem as in the PCRmethod. For LCR, there is also a report on a method of improvingspecificity by adding the step of providing a gap between adjacentprobes and filling the gap by a DNA polymerase. However, what can beexpected in this modified method is specificity only, and there stillremains a problem in that control of temperature is required.Furthermore, use of the additional enzyme leads to an increase in cost.

[0006] A method called the SDA method (strand displacementamplification) [Proc. Natl. Acad. Sci. USA, 89, 392-396, 1992] [NucleicAcid. Res., 20, 1691-1696, 1992] is also known as a method of amplifyingDNA having a sequence complementary to a target sequence as a template.In the SDA method, a special DNA polymerase is used to synthesize acomplementary chain starting from a primer complementary to the 3′-sideof a certain nucleotide sequence while displacing a double-strandedchain if any at the 5′-side of the sequence. In the presentspecification, the simple expression “N5′-side” or “3′-side” refers tothat of a chain serving as a template. Because a double-stranded chainat the 5′-side is displaced by a newly synthesized complementary chain,this technique is called the SDA method. The temperature-changing stepessential in the PCR method can be eliminated in the SDA method bypreviously inserting a restriction enzyme recognition sequence into anannealed sequence as a primer. That is, a nick generated by arestriction enzyme gives a 3′-OH group acting as the origin of synthesisof complementary chain, and the previously synthesized complementarychain is released as a single-stranded chain by strand displacementsynthesis and then utilized again as a template for subsequent synthesisof complementary chain. In this manner, the complicated control oftemperature essential in the PCR method is not required in the SDAmethod.

[0007] In the SDA method, however, the restriction enzyme generating anick should be used in addition to the strand displacement-type DNApolymerase. This requirement for the additional enzyme is a major causefor higher cost. Further, because the restriction enzyme is to be usednot for cleavage of both double-stranded chains but for introduction ofa nick (that is, cleavage of only one of the chains), a dNTP derivativesuch as α-thio dNTP should be used as a substrate for synthesis torender the other chain resistant to digestion with the enzyme.Accordingly, the amplification product by SDA has a different structurefrom that of natural nucleic acid, and there is a limit to cleavage withrestriction enzymes or application of the amplification product to genecloning. In this respect too, there is a major cause for higher cost. Inaddition, when the SDA method is applied to an unknown sequence, thereis the possibility that the same nucleotide sequence as the restrictionenzyme recognition sequence used for introducing a nick may be presentin a region to be synthesized. In this case, it is possible that acomplete complementary chain is prevented from being synthesized.

[0008] NASBA (nucleic acid sequence-based amplification, also called theTMA/transcription mediated amplification method) is known as a method ofamplifying nucleic acid wherein the complicated control of temperatureis not necessary. NASBA is a reaction system wherein DNA is synthesizedby DNA polymerase in the presence of target RNA as a template with aprobe having a T7 promoter added thereto, and the product is formed witha second probe into a double-stranded chain, followed by transcriptionby T7 RNA polymerase with the formed double-stranded chain as a templateto amplify a large amount of RNA (Nature, 350, 91-92, 1991). NASBArequires some heat denaturation steps until double-stranded DNA iscompleted, but the subsequent transcriptional reaction by T7 RNApolymerase proceeds under isothermal conditions. However, a combinationof plural enzymes such as reverse transcriptase, RNase H, DNA polymeraseand T7 RNA polymerase is essential, and this is unfavorable for costsimilarly to SDA. Further, because it is complicated to set upconditions for a plurality of enzyme reaction, this method is hardlywidespread as a general analytical method. In the known reactions ofamplification of nucleic acid, there remain problems such as complicatedcontrol of temperature and the necessity for plural enzymes as describedabove

[0009] For these known reactions of synthesizing nucleic acid, there arefew reports on an attempt for further improving the efficiency ofsynthesis of nucleic acid without sacrificing specificity or cost. Forexample, in a method called RCA (rolling-circle amplification), it wasshown that single-stranded DNA having a series of nucleotide sequencescomplementary to a padlock probe can be synthesized continuously in thepresence of a target nucleotide sequence (Paul M. Lizardi et al., NatureGenetics, 12, 225-232, July, 1998). In RCA, a padlock probe having aspecial structure wherein each of the 5′- and 3′-terminals of a singleoligonucleotide constitutes an adjacent probe in LCR is utilized. Then,the continuous reaction of synthesizing complementary chain with thepadlock probe as a template which was ligated and cyclized in thepresence of a target nucleotide sequence is triggered by combinationwith a polymerase catalyzing the strand displacement-type reaction ofsynthesizing complementary chain. Single-stranded nucleic acid having astructure of a series of regions each consisting of the same nucleotidesequence is thus formed. A primer is further annealed to thissingle-stranded nucleic acid to synthesize its complementary chain and ahigh degree of amplification is thus realized. However, there stillremains the problem of the necessity for a plurality of enzymes.Further, triggering of synthesis of complementary chain depends on thereaction of ligating two adjacent regions, and its specificity isbasically the same as in LCR.

[0010] For the object of supplying 3′-OH, there is a known method inwhich a nucleotide sequence is provided at the 3′-terminal with asequence complementary thereto and a hair pin loop is formed at theterminal (Gene, 71, 29-40, 1988). Synthesis of complementary chain witha target sequence itself as a template starts at the hairpin loop toform single-stranded nucleic acid composed of the complementarynucleotide sequence. For example, a structure in which annealing occursin the same chain at the terminal to which a complementary nucleotidesequence has been linked is realized in PCT/FR95/00891. In this method,however, the step in which the terminal cancels base pairing with thecomplementary chain and base pairing is constituted again in the samechain is essential. It is estimated that this step proceeds depending ona subtle equilibrium state at the terminal of mutually complementarynucleotide sequences involving base pairing. That is, an equilibriumstate maintained between base pairing with a complementary chain andbase pairing in the same chain is utilized and the only chain annealingto the nucleotide sequence in the same chain serves as the origin ofsynthesis of a complementary chain. Accordingly, it is considered thatstrict reaction conditions should be set to achieve high reactionefficiency. Further, in this prior art, the primer itself forms a loopstructure. Accordingly, once a primer dimer is formed, amplificationreaction is automatically initiated regardless of whether a targetnucleotide sequence is present or not, and an unspecific syntheticproduct is thus formed. This can be a serious problem. Further,formation of the primer dimer and subsequent consumption of the primerby unspecific synthetic reaction lead to a reduction in theamplification efficiency of the desired reaction.

[0011] Besides, there is a report that a region not serving as atemplate for DNA polymerase was utilized to realize a 3′-terminalstructure annealing to the same chain (EP713922). This report also hasthe same problem as in PCT/FR95/00891 supra in respect of theutilization of dynamic equilibrium at the terminal or the possibility ofunspecific synthetic reaction due to formation of a dimer primer.Further, a special region not serving as a template for DNA polymeraseshould be prepared as a primer.

[0012] Further, in various signal amplification reactions to which theprinciple of NASBA described above is applied, an oligonucleotide havinga hairpin structure at the terminal thereof is often utilized to supplya double-stranded promoter region (JP-A 5-211873). However, thesetechniques are not those permitting successive supply of 3′-OH forsynthesis of a complementary chain. Further, a hairpin loop structurehaving a 3′-terminal annealed in the same chain is utilized for thepurpose of obtaining a DNA template transcribed by RNA polymerase isutilized in JP-A10-510161 (WO96/17079). In this method, the template isamplified by using transcription into RNA and reverse transcription fromRNA to DNA. In this method, however, the reaction system cannot beconstituted without a combination of a plurality of enzymes.

DISCLOSURE OF THE INVENTION

[0013] The object of the present invention is to provide a method ofsynthesizing nucleic acid based on a novel principle. A more specificobject is to provide a method capable of realizing the synthesis ofnucleic acid depending on sequence efficiently at low costs. That is, anobject of the present invention is to provide a method capable ofachieving the synthesis and amplification of nucleic acid by a singleenzyme even under isothermal reaction conditions. Another object of thepresent invention is to provide a method of synthesizing nucleic acidwhich can realize high specificity difficult to achieve in the knownreaction principle of nucleic acid synthesis, as well as a method ofamplifying nucleic acid by applying said synthetic method.

[0014] The present inventors focused their attention on the fact thatthe utilization of a polymerase catalyzing strand displacement-typesynthesis of complementary chain is useful for nucleic acid synthesisnot depending on complicated control of temperature. Such a DNApolymerase is an enzyme utilized in SDA and RCA. However, even if suchan enzyme is used, another enzyme reaction is always required forsupplying 3′-OH as the origin of synthesis in the known means based onprimers, such as SDA.

[0015] Under these circumstances, the present inventors examined supplyof 3′-OH from a completely different viewpoint from the known approach.As a result, the present inventors found that by utilizing anoligonucleotide having a special structure, 3′-OH can be suppliedwithout any additional enzyme reaction, thereby completing the presentinvention. That is, the present invention relates to a method ofsynthesizing nucleic acid, a method of amplifying nucleic acid byapplying said method of synthesizing nucleic acid and a noveloligonucleotide enabling said methods, as follows:

[0016] 1. A method of synthesizing nucleic acid having complementarynucleotide sequences linked alternately in a single-stranded chain,comprising:

[0017] a) the step of giving nucleic acid which is provided at the3′-terminal thereof with a region F1 capable of annealing to a part F1cin the same chain and which upon annealing of the region F1 to F1c, iscapable of forming a loop containing a region F2c capable of basepairing,

[0018] b) the step of performing synthesis of a complementary chainwherein the 3′-terminal of F1 having annealed to F1c serves as theorigin of synthesis,

[0019] c) the step of annealing, to a region F2c, of an oligonucleotideprovided with the 3′-terminal thereof with F2 consisting of a sequencecomplementary to the region F2c followed by synthesis, with saidoligonucleotide as the origin of synthesis, of a complementary chain bya polymerase catalyzing the strand displacement reaction of synthesizinga complementary chain to displace the complementary chain synthesized instep b), and d) the step of annealing, to the complementary chaindisplaced in step c) to be ready for base pairing, of a polynucleotideprovided at the 3′-terminal thereof with a sequence complementary to anarbitrary region in said chain synthesized in step c), followed bysynthesis, with said 3′-terminal as the origin of synthesis, of acomplementary chain by a polymerase catalyzing the strand displacementreaction of synthesizing a complementary chain to displace thecomplementary chain synthesized in step c).

[0020] 2. The method according to item 1, wherein in step d), the originof synthesis is a region R1 present at the 3′-terminal in the same chainand capable of annealing to a region R1c, and a loop containing theregion R2c capable of base pairing is formed by annealing R1 to R1c.

[0021] 3. An oligonucleotide composed of at least two regions X2 and X1cbelow, and X1c is linked to the 5′-side of X2,

[0022] X2: a region having a nucleotide sequence complementary to anarbitrary region X2c in nucleic acid having a specific nucleotidesequence, and

[0023] X1c: a region having substantially the same nucleotide sequenceas in a region X1c located at the 5′-side of the region X2c in nucleicacid having a specific nucleotide sequence.

[0024] 4. The method according to item 1, wherein the nucleic acid instep a) is second nucleic acid provided by the following steps:

[0025] i) the step of annealing, to a region F2c in nucleic acid servingas a template, of a region F2 in the oligonucleotide described in item 3wherein the region X2 is a region F2 and the region X1c is a region F1c,

[0026] ii) the step of synthesizing first nucleic acid having anucleotide sequence complementary to the template wherein F2 in theoligonucleotide serves as the origin of synthesis,

[0027] iii) the step of rendering an arbitrary region in the firstnucleic acid synthesized in step ii) ready for base pairing, and

[0028] iv) the step of annealing an oligonucleotide having a nucleotidesequence complementary to the region made ready for base pairing in thefirst nucleic acid in step iii), followed by synthesizing second nucleicacid with said oligonucleotide as the origin of synthesis and renderingF1 at the 3′-terminal thereof ready for base pairing.

[0029] 5. The method according to item 4, wherein the region enablingbase pairing in step iii) is. R2c, and the oligonucleotide in step iv)is the oligonucleotide described in item 3 wherein the region X2c is aregion R2c and the region X1c is a region R1c.

[0030] 6. The method according to item 4 or 5, wherein the step ofrendering base pairing ready in steps iii) and iv) is conducted by thestrand displacement synthesis of complementary chain by a polymerasecatalyzing the strand displacement reaction of synthesizingcomplementary chain wherein an outer primer annealing to the 3′ side ofF2c in the template and an outer primer annealing to the 3′-side of theregion used as the origin of synthesis in step iv) for the first nucleicacid serve as the origin of synthesis.

[0031] 7. The method according to item 6, wherein the meltingtemperature of each oligonucleotide and its complementary region in thetemplate used in the reaction is in the following relationship under thesame stringency:

(outer primer/region at the 3′-side in the template)≦(F2c/F2 andR2c/R2)≦(F1c/F1 and R1c/R1).

[0032] 8. The method according to any one of items 4 to 7, wherein thenucleic acid serving as the template is RNA, and the synthesis ofcomplementary chain in step ii) is conducted by an enzyme having areverse transcriptase activity. 9. A method of amplifying nucleic acidhaving complementary nucleotide sequences linked alternately in asingle-stranded chain by repeatedly conducting the following steps:

[0033] A) the step of providing a template which is provided at the 3′-and 5′-terminals thereof with a region consisting of a nucleotidesequence complementary to each terminal region in the same chain andwhich upon annealing of these mutually complementary nucleotidesequences, forms a loop capable of base pairing therebetween,

[0034] B) the step of performing the synthesis of complementary chainwherein the 3′-terminal of said template annealed to the same chainserves as the origin of synthesis,

[0035] C) the step of annealing, to the loop portion, of anoligonucleotide provided at the 3′-terminal thereof with a complementarynucleotide sequence to a loop which among said loops, is located at the3′-terminal site, followed by synthesis, with the oligonucleotide as theorigin of synthesis, of a complementary chain by a polymerase catalyzingthe strand displacement reaction of synthesizing a complementary chainto displace the complementary chain synthesized in step B) to make the3′-terminal thereof ready for base pairing, and

[0036] D) the step wherein the chain with the 3′-terminal made ready forbase pairing in step C) serves as a new template.

[0037] 10. The method according to item 9, wherein the oligonucleotidein step C) is provided at the 5′-terminal thereof with a nucleotidesequence complementary to the 3′-terminal serving as the origin ofsynthesis in step B).

[0038] 11. The method according to item 10, further comprising the stepwhere a complementary, chain synthesized with the oligonucleotide instep C) as the origin of synthesis is used as a template in step A).

[0039] 12. The method according to item 9, wherein the template in stepA) is synthesized by the method described in item 5.

[0040] 13. The method according to item 1 or 9, wherein the stranddisplacement reaction of synthesizing complementary chain is carried outin the presence of a melting temperature regulator.

[0041] 14. The method according to item 13, wherein the meltingtemperature regulator is betaine.

[0042] 15. The method according to item 14, wherein 0.2 to 3.0 M betaineis allowed to be present in the reaction solution.

[0043] 16. A method of detecting a target nucleotide sequence in asample, which comprises performing an amplification method described inany one of items 9 to 15 and observing whether an amplification reactionproduct is generated or not.

[0044] 17. The method according to item 16, wherein a probe containing anucleotide sequence complementary to the loop is added to theamplification reaction product and hybridization therebetween isobserved.

[0045] 18. The method according to item 17, wherein the probe is labeledon particles and aggregation reaction occurring upon hybridization isobserved.

[0046] 19. The method according to item 16, wherein an amplificationmethod described in any one of items 9 to 15 is conducted in thepresence of a detector for nucleic acid, and whether an amplificationreaction product is generated or not is observed on the basis of achange in the signal of the detector.

[0047] 20. A method of detecting a mutation in a target nucleotidesequence by the detection method described in item 16, wherein amutation in a nucleotide sequence as the subject of amplificationprevents synthesis of any one of complementary chains constituting theamplification method.

[0048] 21. A kit for synthesis of nucleic acid having complementarychains alternately linked in a single-stranded chain, comprising thefollowing elements:

[0049] i) the oligonucleotide described in item 3 wherein the region F2cin nucleic acid as a template is X2c, and F1c located at the 5′-side ofF2c is X1c;

[0050] ii) an oligonucleotide containing a nucleotide sequencecomplementary to an arbitrary region in a complementary chainsynthesized with the oligonucleotide in (i) as a primer;

[0051] iii) an oligonucleotide having a nucleotide sequencecomplementary to a region F3c located at the 3′-side of the region F2cin the nucleic acid serving as a template;

[0052] iv) a DNA polymerase catalyzing the strand displacement-typereaction of synthesizing complementary chain; and

[0053] v) a nucleotide serving as a substrate for the element iv).

[0054] 22. The kit according to item 21, wherein the oligonucleotide inii) is the oligonucleotide described in item 3 wherein an arbitraryregion R2c in a complementary chain synthesized with the oligonucleotidein i) as the origin of synthesis is X2c, and R1c located at the 5′ ofR2c is X1c.

[0055] 23. The kit according to item 22, further comprising:

[0056] vi) an oligonucleotide having a nucleotide sequence complementaryto a region R3c located at the 3′-side of the arbitrary R2c in acomplementary chain synthesized with the oligonucleotide in i) as theorigin of synthesis.

[0057] 24. A kit for detection of a target nucleotide sequence,comprising a detector for detection of a product of nucleic acidsynthetic reaction additionally in a kit described in any one of items21 to 23.

[0058] The nucleic acid having complementary nucleotide sequences linkedalternately in a single-stranded chain as the object of synthesis in thepresent invention means nucleic acid having mutually complementarynucleotide sequences linked side by side in a single-stranded chain.Further, in the present invention, it should contain a nucleotidesequence for forming a loop between the complementary chains. In thepresent invention, this sequence is called the loop-forming sequence.The nucleic acid synthesized by the present invention is composedsubstantially of mutually complementary chains linked via theloop-forming sequence. In general, a strand not separated into 2 or moremolecules upon dissociation of base pairing is called a single-strandedchain regardless of whether it partially involves base pairing or not.The complementary nucleotide sequence can form base pairing in the samechain. An intramolecular base-paired product, which can be obtained bypermitting the nucleic acid having complementary nucleotide sequenceslinked alternately in a single-stranded chain according to the presentinvention to be base-paired in the same chain, gives a regionconstituting an apparently double-stranded chain and a loop notinvolving base pairing.

[0059] That is, the nucleic acid having complementary nucleotidesequences linked alternately in a single-stranded chain according to thepresent invention contains complementary nucleotide sequences capable ofannealing in the same chain, and its annealed product can be defined assingle-stranded nucleic acid constituting a loop not involving basepairing at a bent hinged portion. A nucleotide having a nucleotidesequence complementary thereto can anneal to the loop not involving basepairing. The loop-forming sequence can be an arbitrary nucleotidesequence. The loop-forming sequence is capable of base pairing so as toinitiate the synthesis of a complementary chain for displacement, and isprovided preferably with a sequence distinguishable from a nucleotidesequence located in the other region in order to achieve specificannealing. For example, in a preferred embodiment, the loop-formingsequence contains substantially the same nucleotide sequence as a regionF2c (or R2c) located at the 3′-side of a region (i.e. F1c or R1c)derived from nucleic acid as a template and annealed in the same chain.

[0060] In the present invention, substantially the same nucleotidesequence is defined as follows. That is, when a complementary chainsynthesized with a certain sequence as a template anneals to a targetnucleotide sequence to give the origin of synthesizing a complementarychain, this certain sequence is substantially the same as the targetnucleotide sequence. For example, substantially the same sequence as F2includes not only absolutely the same nucleotide sequence as F2 but alsoa nucleotide sequence capable of functioning as a template giving anucleotide sequence capable of annealing to F2 and acting as the originof synthesizing complementary chain. The term “anneal” in the presentinvention means formation of a double-stranded structure of nucleic acidthrough base pairing based on the law of Watson-Crick. Accordingly, evenif a nucleic acid chain constituting base pairing is a single-strandedchain, annealing occurs if intramolecular complementary nucleotidesequences are base-paired. In the present invention, annealing andhybridization have the same meaning in that the nucleic acid constitutesa double-stranded structure through base pairing.

[0061] The number of pairs of complementary nucleotide sequencesconstituting the nucleic acid according to the present invention is atleast 1. According to a desired mode of the present invention, it may be2 or more. In this case, there is theoretically no upper limit to thenumber of pairs of complementary nucleotide sequences constituting thenucleic acid. When the nucleic acid as the synthetic product of thepresent invention is constituted of plural sets of complementarynucleotide sequences, this nucleic acid is composed of repeatedidentical nucleotide sequences.

[0062] The nucleic acid having complementary nucleotide sequences linkedalternately in a single-stranded chain synthesized by the presentinvention may not have the same structure as naturally occurring nucleicacid. It is known that if a nucleotide derivative is used as a substratewhen nucleic acid is synthesized by the action of a DNA polymerase, anucleic acid derivative can be synthesized. The nucleotide derivativeused includes nucleotides labeled with a radioisotope or nucleotidederivatives labeled with a binding ligand such as biotin or digoxin.These nucleotide derivatives can be used to label nucleic acidderivatives as the product. Alternatively, if fluorescent nucleotidesare used as a substrate, the nucleic acid as the product can be afluorescent derivative. Further, this product may be either DNA or RNA.Which one is formed is determined by a combination of the structure of aprimer, the type of a substrate for polymerization and polymerizationreagents for carrying out polymerization of nucleic acid.

[0063] Synthesis of the nucleic acid having the structure describedabove can be initiated by use of a DNA polymerase having the stranddisplacement activity and nucleic acid which is provided at the3′-terminal thereof with a region F1 capable of annealing to a part F1cin the same chain and which upon annealing of the region F1 to F1c, iscapable of forming a loop containing a region F2c capable of basepairing. There are many reports on the reaction of synthesizingcomplementary chain wherein a hairpin loop is formed and a samplesequence itself is used as a template, while in the present inventionthe portion of the hairpin loop is provided with a region capable ofbase pairing, and there is a novel feature on utilization of this regionin synthesizing complementary chain. By use of this region as the originof synthesis, a complementary chain previously synthesized with a samplesequence itself as a template is displaced. Then, a region R1c(arbitrary region) located at the 3′-terminal of the displaced chain isin a state ready for base-pairing. A region having a complementarysequence to this R1c is annealed thereto, resulting information of thenucleic acid (2 molecules) having a nucleotide sequence extending fromF1 to R1c and its complementary chain linked alternately via theloop-forming sequence. In the present invention,, the arbitrary regionsuch as R1c above can be selected arbitrarily provided that it can beannealed to a polynucleotide having a nucleotide sequence complementaryto that region, and that a complementary chain synthesized with thepolynucleotide as the origin of synthesis has necessary functions forthe present invention.

[0064] In the present invention, the term “nucleic acid” is used. Thenucleic acid in the present invention generally includes both DNA andRNA. However, nucleic acid whose nucleotide is replaced by an artificialderivative or modified nucleic acid from natural DNA or RNA is alsoincluded in the nucleic acid of the present invention insofar as itfunctions as a template for synthesis of complementary chain. Thenucleic acid of the present invention is generally contained in abiological sample. The biological sample includes animal, plant ormicrobial tissues, cells, cultures and excretions, or extractstherefrom. The biological sample of the present invention includesintracellular parasitic genomic DNA or RNA such as virus or mycoplasma.The nucleic acid of the present invention may be derived from nucleicacid contained in said biological sample. For example, cDNA synthesizedfrom mRNA, or nucleic acid amplified on the basis of nucleic acidderived from the biological sample, is a typical example of the nucleicacid of the present invention.

[0065] The nucleic acid characteristic of the present invention which isprovided at the 3′-terminal thereof with a region F1 capable ofannealing to a part F1c in the same chain and which upon annealing ofthe region F1 to F1c, is capable of forming a loop containing a regionF2c capable of base pairing can be obtained in various methods. In themost preferable embodiment, the reaction of synthesizing complementarychain utilizing an oligonucleotide having the following structure can beused to give the structure.

[0066] That is, the useful oligonucleotide in the present inventionconsists of at least two regions X2 and X1c below wherein X1c is ligatedto the 5′-side of X2.

[0067] X2: a region having a nucleotide sequence complementary to aregion X2c in nucleic acid having a specific nucleotide sequence.

[0068] X1c: a region having substantially the same nucleotide sequenceas a region X1c located at the 5′-side of the region X2c in nucleic acidhaving a specific nucleotide sequence.

[0069] Here, the nucleic acid having a specific nucleotide sequence bywhich the structure of the oligonucleotide of the invention isdetermined refers to nucleic acid serving as a template when theoligonucleotide of the present invention is used as a primer. In thecase of detection of nucleic acid based on the synthetic method of thepresent invention, the nucleic acid having a specific nucleotidesequence is a detection target or nucleic acid derived from thedetection target. The nucleic acid having a specific nucleotide sequencerefers to nucleic acid wherein at least a part of the nucleotidesequence is revealed or predictable. The part of the nucleotide sequencerevealed is the region X2c and the region X1c located at the 5′-sidethereof. It can be supposed that these 2 regions are contiguous orlocated apart from each other. By the relative positional relationshipof the two, the state of a loop formed upon self-annealing of nucleicacid as the product is determined. The distance between the two ispreferably not very apart from each other in order that nucleic acid asthe product is subjected to self-annealing preferentially overintermolecular annealing. Accordingly, the positional relationship ofthe two is preferably that they are contiguous via a distance of usually0 to 100 bases. However, in the formation of a loop by self-annealingdescribed below, there can be the case where it would be disadvantageousfor formation of a loop in a desired state that the two are too close toeach other. In the loop, there is a need for a structure for annealingof a new oligonucleotide and for readily initiating thestrand-displacement reaction of synthesizing a complementary chain withsaid oligonucleotide as the origin of synthesis. More preferably, thedistance between the region X2c and the region X1c located at the5′-side of X2c is designed to be 0 to 100 bases, more desirably 10 to 70bases. This numerical value shows a length excluding X1c and X2. Thenumber of bases constituting the part of a loop is that of this lengthplus a region corresponding to X2.

[0070] Both the terms “same” and “complementary” used forcharacterization of the nucleotide sequence constituting theoligonucleotide based on the present invention do not mean beingabsolutely the same or absolutely complementary. That is, the samesequence as a certain sequence includes sequences complementary tonucleotide sequences capable of annealing to a certain sequence. On theother hand, the complementary sequence means a sequence capable ofannealing under stringent conditions to provide a 3′-terminal serving asthe origin of synthesis of complementary chain.

[0071] Usually, the regions X2 and X1c constituting the oligonucleotideof the present invention for the nucleic acid having a specificnucleotide sequence are located contiguously without being overlapped.If there is a common part in both the nucleotide sequences, the two canbe partially overlaid. Because X2 should function as a primer, it shouldalways be a 3′-terminal. On the other hand, X1c should give the functionof a primer as described blow to the 3′-terminal of a complementarychain synthesized with the nucleic acid as a template, and thus it shallbe arranged at the 5′-terminal. The complementary chain obtained withthis oligonucleotide as the origin of synthesis serves as a template forsynthesis of complementary chain in the reverse direction in the nextstep, and finally the part of the oligonucleotide of the presentinvention is copied as a template into a complementary chain. The3′-terminal generated by copying has the nucleotide sequence X1, whichanneals to X1c in the same chain to form a loop.

[0072] In the present invention, the oligonucleotide means the one thatsatisfies the 2 requirements, that is, it must be able to formcomplementary base pairing and give an —OH group serving as the originof synthesis of complementary chain at the 3′-terminal. Accordingly, itsbackbone is not necessarily limited to the one via phosphodiesterlinkages. For example, it may be composed of a phosphothioate derivativehaving S in place of P as a backbone or a peptide nucleic acid based onpeptide linkages. The bases may be those capable of complementary basepairing. In the nature, there are 5 bases, that is, A, C, T, G and U,but the base can be an analogue such as bromodeoxyuridine. Theoligonucleotide used in the present invention functions preferably notonly as the origin of synthesis but also as a template for synthesis ofcomplementary chain. The term polynucleotide in the present inventionincludes oligonucleotides. The term “polynucleotide” is used in the casewhere the chain length is not limited, while the term “oligonucleotide∞is used to refer to a nucleotide polymer having a relatively short chainlength.

[0073] The oligonucleotide according to the present invention has such achain length as to be capable of base pairing with a complementary chainand to maintain necessary specificity under the given environment in thevarious reactions of synthesizing nucleic acid described below.Specifically, it is composed of 5 to 200 base pairs, more preferably 10to 50 base pairs. The chain length of a primer recognizing the knownpolymerase catalyzing the sequence-dependent nucleic acid syntheticreaction is at least about 5 bases, so the chain length of the annealingpart should be longer than that. In addition, a length of 10 bases ormore is desired statistically in order to expect specificity as thenucleotide sequence. On the other hand, preparation of a too longnucleotide sequence by chemical synthesis is difficult, and thus thechain length described above is exemplified as a desired range. Thechain length exemplified here refers to the chain length of a partannealing to a complementary chain. As described below, theoligonucleotide according to the present invention can anneal finally toat least 2 regions individually. Accordingly, it should be understoodthat the chain length exemplified here is the chain length of eachregion constituting the oligonucleotide.

[0074] Further, the oligonucleotide according to the present inventioncan be labeled with a known labeling substance. The labeling substanceincludes binding ligands such as digoxin and biotin, enzymes,fluorescent substances and luminescent substances, and radioisotopes.The techniques of replacing a base constituting an oligonucleotide by afluorescent analogue are also known (WO95/05391, Proc. Natl. Acad. Sci.USA, 91, 6644-6648, 1994).

[0075] Other oligonucleotides according to the present invention canalso have been bound to a solid phase. Alternatively, an arbitrary partof the oligonucleotide may be labeled with a binding ligand such asbiotin, and it can be immobilized indirectly via a binding partner suchas immobilized avidin. When the immobilized oligonucleotide is used asthe origin of synthesis, nucleic acid as the synthetic reaction productis captured by the solid phase, thus facilitating its separation. Theseparated product can be detected by a nucleic acid-specific indicatoror by hybridization with a labeling probe. The target nucleic acidfragments can also be recovered by digesting the product with arbitraryrestriction enzymes.

[0076] The term “template” used in the present invention means nucleicacid serving as a template for synthesizing a complementary chain. Acomplementary chain having a nucleotide sequence complementary to thetemplate has a meaning as a chain corresponding to the template, but therelationship between the two is merely relative. That is, a chainsynthesized as the complementary chain can function again as a template.That is, the complementary chain can become a template.

[0077] The oligonucleotide useful in the present invention is notlimited to the 2 regions described above and can contain an additionalregion. While X2 and X1c are arranged at the 3′- and 5′-terminalsrespectively, an arbitrary sequence can be interposed therebetween. Forexample, it can be a restriction enzyme recognition site, a promoterrecognized by RNA polymerase, or DNA coding for ribozyme. By using it asa restriction enzyme recognition sequence, the nucleic acid having acomplementary sequence alternately linked in a single-stranded chain asthe synthetic product of the present invention can be cleaved intodouble-stranded nucleic acids of the same length. By arranging apromoter sequence recognized by RNA polymerase, the synthetic product ofthe present invention serves as the template to permit furthertranscription into RNA. By further arranging DNA coding for ribozyme, asystem where the transcriptional product is self-cleaved is realized.These additional nucleotide sequences are those functioning after formedinto a double-stranded chain. Accordingly, when the single-strandednucleic acid according to the present invention has formed a loop, thesesequences do not function. They do not function until the nucleic acidis elongated and annealed in the absence of a loop to a chain having acomplementary nucleotide sequence.

[0078] When a promoter is combined with the oligonucleotide based on thepresent invention in such a direction as to permit transcription of thesynthesized region, the reaction product based on the present inventionwhere the same nucleotide sequence is repeated realizes a highlyefficient transcriptional system. By combining this system with asuitable expression system, translation into a protein is also feasible.That is, the system can be utilized for transcription and translationinto protein in bacteria or animal cells or in vitro.

[0079] The oligonucleotide of the present invention having the structuredescribed above can be chemically synthesized. Alternatively, naturalnucleic acid may be cleaved with e.g. restriction enzymes and modifiedso as to be composed of, or ligated into, the nucleotide sequencedescribed above.

[0080] The basic principle of the reaction for performing synthesis byutilizing the useful oligonucleotide described above in combination withDNA polymerase having the strand displacement activity in the reactionof synthesizing nucleic acid according to the present invention isdescribed by reference to FIGS. 5 to 6. The oligonucleotide describedabove (FA in FIG. 5) anneals at X2 (corresponding to F2) to nucleic acidas a template, to provide the origin of synthesis of complementarychain. In FIG. 5, a complementary chain synthesized from FA as theorigin of synthesis is displaced by synthesis of complementary chain(described below) from an outer primer (F3), to form a single-strandedchain (FIG. 5-A). When synthesis of complementary chain to the resultingcomplementary chain is further conducted, the 3′-terminal of nucleicacid synthesized as complementary chain in FIG. 5-A has a nucleotidesequence complementary to the oligonucleotide of the present invention.That is, because the 5′-terminal of the oligonucleotide of the presentinvention has the same sequence as a region X1c (corresponding to F1c),the 3′-terminal of the nucleic acid thus synthesized has a complementarysequence X1 (F1). FIG. 5 shows that the complementary chain synthesizedfrom R1 as the origin of synthesis is displaced by synthesis ofcomplementary chain by primer R3 as the origin of synthesis. Once the3′-terminal portion is made ready for base pairing by this displacement,X1 (F1) at the 3′-terminal anneals to X1c (F1c) in the same chain, andelongation reaction with itself as a template proceeds (FIG. 5-B). Then,X2c (F2c) located at the 3′-terminal thereof is left as a loop notinvolving base pairing. X2 (F2) in the oligonucleotide according to thepresent invention anneals to this loop, and a complementary chain issynthesized with said oligonucleotide as the origin of synthesis (FIG.5-B). A product of complementary chain synthetic reaction with thepreviously synthesized product as a template is displaced by the stranddisplacement reaction so that it is made ready for base pairing.

[0081] By the basic constitution using one kind of oligonucleotideaccording to the present invention and an arbitrary reverse primercapable of conducting nucleic acid synthesis where a complementary chainsynthesized with said oligonucleotide as a primer is used as a template,a plurality of nucleic acid synthetic products as shown in FIG. 6 can beobtained. As can be seen from FIG. 6,(D) is the desired nucleic acidproduct of the invention having complementary nucleotide sequencealternately linked in a single-stranded chain. Once converted into asingle-stranded chain by treatment such as heat denaturation, the otherproduct (E) serves again as a template for forming (D). If the product(D) as nucleic acid in the form of a double-stranded chain is convertedinto a single-stranded chain by heat denaturation, annealing occurswithin the same chain at high probability without forming the originaldouble-stranded chain. This is because a complementary chain having thesame melting temperature (Tm) undergoes intramolecular reactionpreferentially over intermolecular reaction. Each single-stranded chainderived from the product (D) annealed in the same chain is annealed inthe same chain and returned to the state of (B), and each chain furthergives one molecule of (D) and (E) respectively. By repeating thesesteps, it is possible to successively synthesize the nucleic acid havingcomplementary nucleotide sequences linked alternately in asingle-stranded chain. The template and the product formed in 1 cycleare increased exponentially, thus making the reaction very efficient.

[0082] To realize the state of FIG. 5(A), the initially synthesizedcomplementary chain should, in at least the portion to which the reverseprimer anneals, should be ready for base pairing. This step can beachieved by an arbitrary method. That is, an outer primer (F3), whichanneals to the first template at a region F3c at the 3′-side of theregion F2c to which the oligonucleotide of the present inventionanneals, is separately prepared. If this outer primer is used as theorigin synthesis to synthesize a complementary chain by a polymerasecatalyzing the strand displacement-type synthesis of complementarychain, the complementary chain synthesized from the F2c as the origin ofsynthesis in the invention is displaced, and as a result the region R1cto be annealed by R1 is made ready for base pairing (FIG. 5). Byutilization of the strand displacement reaction, the reaction up to nowcan proceed under isothermal conditions.

[0083] When an outer primer is used, synthesis from the outer primer(F3) should be initiated after synthesis from F2c. In the most simplemethod, the concentration of the inner primer is made higher than theconcentration of the outer primer. Specifically, the primers are used atusually 2- to 50-fold, preferably 4- to 10-fold differentconcentrations, whereby the reaction can proceed as expected. Further,the melting temperature (Tm) of the outer primer is set to be lower thanthe Tm of the X1 (corresponding to F1 and R1) in the inner primerwhereby the timing of synthesis can be controlled. That is, (outerprimer F3: F3c)≦F2c/F2)≦(F1c/F1) or (outer primer/region at the 3′-sidein the template)≦(X2c: X2)≦(X1c: X1). Here, the reason for(F2c/F2)≦(F1c/F1) is for annealing between F1c/F1 prior to annealing ofF2 to the loop. The annealing between F1c/F1 is an intramolecularreaction and may thus proceed preferentially at high probability.However, it is meaningful to consider Tm in order to give more desiredreaction conditions. As a matter of course, similar conditions should beconsidered even in the design of a reverse primer. By using such arelationship, statistically ideal reaction conditions can be achieved.If other conditions are fixed, melting temperature (Tm) can betheoretically calculated by a combination of the length of an annealingcomplementary chain and bases constituting base-pairing. Accordingly,those skilled in the art can derive preferable conditions on the basisof the disclosure of this specification.

[0084] Further, the phenomenon called contiguous stacking can also beapplied for controlling timing of annealing of the outer primer.Contiguous stacking is a phenomenon in which an oligonucleotide notcapable of annealing independently is made capable of annealing uponbeing contiguous to the part of a double-stranded chain (ChiaraBorghesi-Nicoletti et al., Bio Techniques, 12, 474-477 (1992)). That is,the outer primer is designed so as to be contiguous to F2c (X2c) and notto be able to anneal independently. By doing so, annealing of the outerprimer does not occur until F2c (X2c) anneals, and thus the annealing ofF2c (X2c) occurs preferentially. On the basis of this principle, theExamples show setting of the nucleotide sequence of an oligonucleotidenecessary as a primer for a series of reactions. This step can also beachieved by denaturation under heating or with a DNA helicase.

[0085] If the template nucleic acid having F2c (X2c) is RNA, the stateof FIG. 5-(A) can also be realized by a different method. For example,if this RNA chain is decomposed, R1 is made ready for base pairing. Thatis, F2 is annealed to F2c in RNA and a complementary chain issynthesized as DNA by a reverse transcriptase. The RNA serving as atemplate is decomposed by alkali denaturation or by enzymatic treatmentwith a ribonuclease acting on RNA in a double-stranded chain of DNA/RNAwhereby the DNA synthesized from F2 is formed into a single-strandedchain. For the enzyme selectively decomposing RNA in a double-strandedchain of DNA/RNA, the ribonuclease activity of RNase H or some reversetranscriptases can be utilized. In this manner, the reverse primer canbe annealed to R1c made capable of base pairing. Accordingly, the outerprimer for rendering R1c ready for base pairing becomes unnecessary.

[0086] Alternatively, the strand displacement activity of reversetranscriptase can be utilized for the strand displacement by an outerprimer as described above. In this case, a reaction system can beconstituted by a reverse transcriptase only. That is, using RNA as atemplate, it is made possible by a reverse transcriptase to synthesize acomplementary chain from F2 annealing to F2c in the template and tosynthesize a complementary chain from the outer primer F3 as the originof synthesis annealing to F3c located at the 3′-side of F2c and tosimultaneously displace the previously synthesized complementary chain.When the reverse transcriptase performs the reaction of synthesizing acomplementary chain with DNA as the template, all the reactions ofsynthesizing complementary chains including the synthesis of acomplementary chain with R1 as the origin of synthesis annealing to R1cin the displaced complementary chain as the template, the synthesis of acomplementary chain with R3 as the origin of synthesis annealing to R3clocated at the 3′-side of R1c and the simultaneous displacementreaction, proceed by the reverse transcriptase. If it is not possible toexpect that the reverse transcriptase exhibits the DNA/RNA stranddisplacement activity under given reaction conditions, a DNA polymerasehaving the strand displacement activity described above may be combined.The mode of obtaining a first single-stranded nucleic acid with RNA as atemplate as described above constitutes a preferable mode of the presentinvention. On the other hand, if a DNA polymerase such as Bca DNApolymerase having both strand displacement activity and reversetranscriptase activity is used, not only synthesis of a firstsingle-stranded nucleic acid from RNA but also subsequent reaction withDNA as a template can proceed similarly by the same enzyme.

[0087] The reaction system described above brings about variousvariations inherent in the present invention by utilization of thereverse primer having a specific structure. The most effective variationis described below. That is, the oligonucleotide constituted asdescribed in [5] is used as the reverse primer in the most advantageousmode of the present invention. The oligonucleotide in [5] is anoligonucleotide wherein arbitrary regions R2c and R1c in a complementarychain synthesized with F2 as a primer are X2c and X1c respectively. Byuse of such a reverse primer, a series of reactions for forming a loopand for synthesizing and displacing a complementary chain from this loopoccur in both the sense and antisense chains (forward side and reverseside). As a result, the reaction efficiency for synthesis of the nucleicacid having complementary nucleotide sequences linked alternately in asingle-stranded chain according to the present invention is greatlyimproved while a series of these reactions are feasible under isothermalconditions. Hereinafter, this mode is described in more detail byreference to FIGS. 1 to 3 where this mode is summarized.

[0088] In the following mode, 2 kinds of oligonucleotides based on thepresent invention are prepared. For explanation, these are designated FAand RA. The regions constituting FA and RA are as follows: X2 X1c FA F2F1c RA R2 R1c

[0089] Here, F2 is a complementary nucleotide sequence to a region F2cin nucleic acid as the template. R2 is a nucleotide sequencecomplementary to an arbitrary region R2c contained in a complementarychain synthesized with F2 as a primer. F1c and R1c are arbitrarynucleotide sequences located downward from F2c and R2c respectively. Thedistance between F2 and R2 may be arbitrary. Even if its length is about1 kbp, sufficient synthesis is feasible under suitable conditions,though depending on the synthetic ability of DNA polymerase to performthe synthesis of a complementary chain. Specifically, when Bst DNApolymerase is used, the desired product is certainly synthesized if thedistance between F2 and R2c is 800 bp, preferably 500 bp or less. In PCRinvolving temperature cycle, the reduction in the enzyme activity by thestress of temperature change is considered to reduce the efficiency ofsynthesis of a long nucleotide sequence. In a preferable mode of thepresent invention, the temperature cycle in the step of amplifyingnucleic acid is not required, and thus the synthesis and amplificationof an even long nucleotide sequence can be certainly achieved.

[0090] First, F2 in FA is annealed to nucleic acid as a template andused as the origin of synthesis of a complementary chain. The subsequentreaction steps until FIG. 1 (4) are the same as in the previouslydescribed basic mode (FIG. 5) in the present invention. The sequenceannealed as F3 in FIG. 1 (2) is the outer primer described above. A DNApolymerase for conducting the strand displacement-type synthesis of acomplementary chain with this primer as the origin of synthesis is usedso that the complementary chain synthesized from FA is displaced andmade ready for base pairing.

[0091] When R2c is made ready for base pairing in (4), RA as a reverseprimer anneals thereto in the combination of R2c/R2. Synthesis of acomplementary chain with this site as the origin of synthesis proceedsuntil the chain reaches F1c at the 5′-terminal of FA. Following thisreaction of synthesizing a complementary chain, the outer primer R3 fordisplacement anneals thereto to synthesize a complementary chain, duringwhich strand displacement also proceeds so that the complementary chainsynthesized from RA as the origin of synthesis is displaced. In thecomplementary chain thus displaced, RA is located at the 5′-side thereofand a sequence complementary to FA is located at the 3′-terminalthereof.

[0092] At the 3′-side of the single-stranded nucleic acid thusdisplaced, there is a sequence F1 complementary to F1c in the samechain. F1 rapidly anneals to F1c in the same molecule to initiatesynthesis of a complementary chain. When the 3′-terminal (F1) anneals toF1c in the same chain, a loop containing F2c is formed. As is alsoevident from FIG. 2-(7), the part of this loop remains ready for basepairing. The oligonucleotide FA of the invention having a nucleotidesequence complementary to F2c anneals to the part of this loop and actsas the origin of synthesis of a complementary chain (7). Synthesis of acomplementary chain from the loop proceeds while the reaction product inthe previously initiated complementary chain synthesis from F1 isdisplaced. As a result, the complementary chain synthesized with itselfas the template is made ready for base pairing again at the 3′-terminal.This 3′-terminal is provided with a region R1 capable of annealing toR1c in the same chain, and the two are annealed preferentially due tothe rapid intramolecular reaction. The same reaction as theabove-described reaction starting from the 3′-terminal synthesized withFA as a template proceeds in this region as well. As a result, thenucleic acid having complementary nucleotide sequences linkedalternately in the same single-stranded chain according to the presentinvention is continued to be extended from R1 as the starting point atthe 3′-terminal by successive synthesis of a complementary chain andsubsequent displacement thereof. Because R2c is always contained in theloop formed by intramolecular annealing of the 3′-terminal R1, theoligonucleotide (RA) provided with R2 anneals to the loop at the3′-terminal in the subsequent reaction.

[0093] When attention is paid to nucleic acid synthesized ascomplementary chain from the oligonucleotide annealing to the loop inthe single-stranded nucleic acid elongated with itself as the template,synthesis of the nucleic acid having complementary nucleotide sequenceslinked alternately in the same single-stranded chain according to thepresent invention also proceeds here. That is, synthesis of acomplementary chain from the loop is completed when it reached RA ine.g. FIG. 2-(7). Then, when the nucleic acid displaced by this nucleicacid synthesis initiates synthesis of complementary chain (FIG. 3-(8)),the reaction reaches the loop which was once the origin of synthesis,and displacement is initiated again. In this manner, the nucleic acidinitiated to be synthesized from the loop is also displaced, and as aresult, the 3′-terminal R1 capable of annealing in the same chain isobtained (FIG. 3-(10)). This 3′-terminal R1 anneals to R1c in the samechain to initiate synthesis of complementary chain. This reaction is thesame as in FIG. 2-(7) except that F is used in place of R. Accordingly,the structure shown in FIG. 3-(10)can function as a new nucleic acidwhich continues self-elongation and new nucleic acid formation.

[0094] The reaction of synthesizing nucleic acid, initiated from thenucleic acid shown in FIG. 3-(10), causes elongation from the3′-terminal F1 as the origin of synthesis, as opposed to the reactiondescribed above. That is, in the present invention, as one nucleic acidis elongated, the reaction of continuing to supply a new nucleic acidinitiating elongation separately proceeds. Further, as the chain iselongated, a plurality of loop-forming sequences are brought about notonly at the terminal but also in the same chain. When these loop-formingsequences are made ready for base pairing by the strand displacementsynthetic reaction, an oligonucleotide anneals thereto to serve as abase for the reaction of forming a new nucleic acid. Further efficientamplification is achieved by the synthetic reaction starting not: onlyat the terminal but also in the chain. The oligonucleotide RA based onthe present invention is combined as the reverse primer as describedabove whereby elongation and subsequent formation of a new nucleic acidoccur. Further, in the present invention, this newly formed nucleic aciditself is elongated and brings about subsequent formation of a newnucleic acid. A series of these reactions continue theoreticallypermanently to achieve very efficient amplification of nucleic acid. Inaddition, the reaction in the present invention can be conducted underisothermal conditions.

[0095] The reaction products thus accumulated possess a structure havinga nucleotide sequence between F1 and R1 and its complementary sequencelinked alternately therein. However, both the terminals of the repeatingunit have a region consisting of the successive nucleotide sequencesF2-F1 (F2c-F1c) and R2-R1 (R2c-R1c). For example, in FIG. 3-(9), thesequences (R2-F2c)-(F1-R2c)-(R1-F1c)-(F2-R2c) are linked in this orderfrom the5′-side. This is because the amplification reaction based on thepresent invention proceeds on the principle that the reaction isinitiated from F2 (or R2) with an oligonucleotide as the origin ofsynthesis and then a complementary chain is elongated by the syntheticreaction from F1 (or R1) with the 3′-terminal as the origin ofsynthesis.

[0096] Here, in the most preferable mode, oligonucleotides FA and RAaccording to the present invention were used as oligonucleotidesannealing to the part of a loop. However, even if these oligonucleotidehaving a limited structure are not used, the amplification reactionaccording to the present invention can be carried out by use of anoligonucleotide capable of initiating the synthesis of a complementarychain from the loop. That is, the elongating 3′-terminal, once displacedby a complementary chain synthesized from the loop, gives the part of aloop again. Because the nucleic acid having complementary nucleotidesequences linked alternately in a single-stranded chain is always usedas a template in the complementary chain synthesis starting from theloop, it is evident that the nucleic acid desired in the presentinvention can be synthesized. However, the nucleic acid thus synthesizedperforms synthesis of a complementary chain by forming a loop afterdisplacement, but there is no 3′-terminal available for subsequentformation of a loop, and thus it cannot function as a new template.Accordingly, the product in this case, unlike nucleic acid initiated tobe synthesized by FA or RA, cannot be expected to be exponentiallyamplified. From this reason, an oligonucleotide having the structure ofFA or RA is useful for highly efficient synthesis of nucleic acid basedon the present invention.

[0097] A series of these reactions proceed by adding the followingcomponents to single-stranded nucleic acid as a template and thenincubating the mixture at such a temperature that the nucleotidesequence constituting FA and RA can form stable base pairing with itscomplementary nucleotide sequence while the enzyme activity can bemaintained.

[0098] 4 kinds of oligonucleotides:

[0099] FA,

[0100] RA,

[0101] outer primer F3, and

[0102] outer primer R3,

[0103] DNA polymerase for performing the strand displacement-typesynthesis of complementary chain,

[0104] an oligonucleotide serving as a substrate for DNA polymerase.

[0105] Accordingly, temperature cycle such as in PCR is not necessary.The stable base pairing referred to herein means a state in which atleast a part of an oligonucleotide present in the reaction system cangive the origin of synthesis of complementary chain. For example, thedesired condition for bringing about stable base pairing is to set lowerthan melting temperature (Tm). Generally, melting temperature (TM) isregarded as the temperature at which 50% of nucleic acids havingmutually complementary nucleotide sequences are base-paired. Setting atmelting temperature (Tm) or less is not an essential condition in thepresent invention, but is one of the reaction conditions to beconsidered for attaining high efficiency of synthesis. If nucleic acidto be used as a template is a double-stranded chain, the nucleic acidshould, in at least a region to which the oligonucleotide anneals, bemade ready for base pairing. For this, heat denaturation is generallyconducted, and this may be conducted only once as pretreatment beforethe reaction is initiated.

[0106] This reaction is conducted in the presence of a buffer givingsuitable pH to the enzyme reaction, salts necessary for annealing or formaintaining the catalytic activity of the enzyme, a protective agent forthe enzyme, and as necessary a regulator for melting temperature (Th).As the buffer, e.g. Tris-HCl having a buffering action in a neutral toweakly alkaline range is used. The pH is adjusted depending on the DNApolymerase used. As the salts, KCl, NaCl, (NH₄)₂SO₄ etc. are suitablyadded to maintain the activity of the enzyme and to regulate the meltingtemperature (Tm) of nucleic acid. The protective agent for the enzymemakes use of bovine serum albumin or sugars. Further, dimethyl sulfoxide(DMSO) or formamide is generally used as the regulator for meltingtemperature (Tm). By use of the regulator for melting temperature (Tm),annealing of the oligonucleotide can be regulated under limitedtemperature conditions. Further, betaine (N,N,N-trimethylglycine) or atetraalkyl ammonium salt is also effective for improving the efficiencyof strand displacement by virtue of its isostabilization. By addingbetaine in an amount of 0.2 to 3.0 M, preferably 0.5 to 1.5 M to thereaction solution, its promoting action on the nucleic acidamplification of the present invention can be expected. Because theseregulators for melting temperature act for lowering melting temperature,those conditions giving suitable stringency and reactivity areempirically determined in consideration of the concentration of salts,reaction temperature etc.

[0107] An important feature in the present invention is that a series ofreactions do not proceed unless the positional relationship of aplurality of regions is maintained. By this feature, unspecificsynthetic reaction accompanied by unspecific synthesis of complementarychain is effectively prevented. That is, even if a certain unspecificreaction occurs, the possibility for the product to serve as a startingmaterial in the subsequent amplification step is minimized. Further, theregulation of the progress of reactions by many regions brings about thepossibility that a detection system capable of strict identification ofthe desired product in analogous nucleotide sequences can be arbitrarilyconstituted.

[0108] This feature can be utilized for detection of mutations in agene. In the mode of the invention where the outer primer is used, 4primers, that is, 2 outer primers and 2 primers consisting of theoligonucleotides of the present invention, are used. That is, unless the6 regions contained in the 4 oligonucleotides work as designed, thesynthetic reaction of the present invention do not proceed. Inparticular, the sequences of the 3′-terminal of each oligonucleotide asthe origin of synthesis of complementary chain and of the 5′-terminal ofthe X1c region where the complementary chain serves as the origin ofsynthesis are important. Hence, these important sequences is designed soas to correspond to a mutation to be detected, and the syntheticreaction product of the present invention is observed whereby thepresence or absence of a mutation such as base deletion or insertion, orgenetic polymorphism such as SNPs can be comprehensively analyzed.Specifically, bases estimated to have a mutation or polymorphism aredesigned so as to correspond to the vicinity of the 3′-terminal of anoligonucleotide as the origin of synthesis of complementary chain, or of5′-terminal thereof when a complementary chain is the origin ofsynthesis. If a mismatch is present at the 3′-terminal as the origin ofsynthesis of complementary chain or in its vicinity, the reaction ofsynthesizing a complementary chain to nucleic acid is significantlyinhibited. In the present invention, a high degree of amplificationreaction is not achieved unless the structure of the terminals of aproduct in the initial reaction brings about repeated reactions.Accordingly, even if erroneous synthesis occurs, complementary chainsynthesis constituting amplification reaction is always interrupted insome of the steps, and thus a high degree of amplification reaction doesnot occur in the presence of a mismatch. As a result, the mismatcheffectively inhibits amplification reaction, and an accurate result isfinally brought about. That is, it can be said that the amplificationreaction of nucleic acid based on the present invention has a highlycompleted mechanism for checking the nucleotide sequence. These featuresare an advantage hardly expectable in e.g. the PCR method whereamplification reaction is performed in mere 2 regions.

[0109] The region X1c characterizing the oligonucleotide used in thepresent invention can serve as the origin of synthesis after acomplementary sequence is synthesized, and this complementary sequenceanneals to the sequence X1 in the same newly synthesized chain wherebysynthetic reaction with itself as a template proceeds. Therefore, evenif the so-called primer dimer which is often problematic in the priorart is formed, this oligonucleotide does not form a loop. Accordingly,unspecific amplification attributable to the primer dimer cannot occurtheoretically, and thus the present oligonucleotide contributes to animprovement in the specificity of the reaction.

[0110] Further, according to the present invention, the outer primersshown as F3 (FIG. 1-(2)) or R3 (FIG. 2-(5)) are combined whereby aseries of the reactions described above can be conducted underisothermal conditions. That is, the present invention provides a methodof amplifying nucleic acid having complementary sequences linkedalternately in a single-stranded chain, which comprises the steps shownin item 9 above. In this method, temperature conditions where stableannealing occurs between F2c/F2, between R2c/R2, between F1c/F1, andbetween R1c/R1 are selected, and preferably F3c/F3 and R3c/R3 are set upto be annealed by the phenomenon of contiguous stacking facilitated byannealing of F2c/F2 and R2c/R2, respectively.

[0111] In the present invention, the terms “synthesis” and“amplification” of nucleic acid are used. The synthesis of nucleic acidin the present invention means the elongation of nucleic acid from anoligonucleotide serving as the origin of synthesis. If not only thissynthesis but also the formation of other nucleic acid and theelongation reaction of this formed nucleic acid occur continuously, aseries of these reactions is comprehensively called amplification.

[0112] The single-stranded nucleic acid which is provided at the3′-terminal thereof with a region F1 capable of annealing to a part F1cin the same chain and which upon annealing of the region F1 to F1c inthe same chain, is capable of forming a loop containing a region F2ccapable of base pairing is an important element of the presentinvention. Such a single-stranded nucleic acid can also be supplied onthe following principle. That is, the synthesis of a complementary chainis allowed to proceed on the basis of a primer having the followingstructure. 5′-[region X1 annealing to region X1c located inprimer]-[loop forming sequence ready for base pairing]-[regionX1c]-[region having a sequence complementary to a template]-3′

[0113] As the region having a sequence complementary, to a template, twonucleotide sequences, that is, a nucleotide sequence (primer FA)complementary to F1 and a nucleotide sequence (primer RA) complementaryto R1c, are prepared. The nucleotide sequence constituting nucleic acidto be synthesized contains a nucleotide sequence extending from theregion F1 to the region R1c and a nucleotide sequence extending from theregion R1 having a nucleotide sequence complementary to this nucleotidesequence to the region F1c. X1c and X1 capable of annealing in theinside of the primer can be arbitrary sequences. However, in a regionbetween primers FA and RF, the sequence of the region X1c/X1 is madepreferably different.

[0114] First, the synthesis of a complementary chain by the primer FAfrom the region F1 in template nucleic acid is conducted. Then, theregion R1c in the synthesized complementary chain is made ready for basepairing, to which the other primer is annealed to form the origin ofsynthesis of complementary chain. The 3′-terminal of the complementarychain synthesized in this step has a nucleotide sequence complementaryto the primer FA constituting the 5′-terminal of the initiallysynthesized chain, so it has been provided at the 3′-terminal thereofwith the region X1 which anneals to the region X1c in the same chain toform a loop. The characteristic 3′-terminal structure according to thepresent invention is thus provided, and the subsequent reactionconstitutes the reaction system shown previously as the most preferablemode. The oligonucleotide annealing to the portion of the loop isprovided at the 3′-terminal thereof with the region X2 complementary tothe region X2c located in the loop and at the 5′-terminal thereof withthe region X1. In the previous reaction system, primers FA and RA wereused to synthesize a chain complementary to template nucleic acidthereby giving a loop structure to the 3′-terminal of the nucleic acid.In this method, the terminal structure characteristic of the presentinvention is provided by the short primers. In this mode, on the otherhand, the whole of a nucleotide sequence constituting a loop is providedas a primer, and synthesis of this longer primer is necessary.

[0115] If a nucleotide sequence containing restriction enzymerecognition regions is used as a reverse primer, a different modeaccording to the present invention can be constituted. The reaction witha reverse primer containing a restriction enzyme recognition sequence isspecifically described by reference to FIG. 6. When FIG. 6-(D) iscompleted, a nick is generated by a restriction enzyme corresponding toa restriction enzyme recognition site in the reverse primer. The stranddisplacement-type reaction of synthesizing complementary chain isinitiated from this nick as the origin of synthesis. Because the reverseprimers are located at both the terminals of a double-stranded nucleicacid constituting (D), the reaction of synthesizing complementary chainis also initiated from both the terminals. Though basically based on theSDA method described as the prior art, the nucleotide sequence servingas a template has a structure having complementary nucleotide sequencesalternately linked according to the present invention so that thenucleic acid synthetic system unique to the present invention isconstituted. A part serving as a complementary chain of the reverseprimer to be nicked should be designed to incorporate a dNTP derivativesuch that it is rendered nuclease resistance to prevent cleavage of thedouble-stranded chain by the restriction enzyme.

[0116] It is also possible to insert a promoter for RNA polymerase intothe reverse primer. Transcription from both the terminals in FIG. 6-(D)is performed by a RNA polymerase recognizing this promoter in this casetoo similar to the previous mode where the SDA method was applied.

[0117] The nucleic acid synthesized in the present invention is asingle-stranded chain but is composed of partial complementarysequences, and thus the majority of these sequences are base-paired. Byuse of this feature, the synthesized product can be detected. Bycarrying out the method of synthesizing nucleic acid according to thepresent invention in the presence of a fluorescent pigment as adouble-stranded chain-specific intercalater such as ethidium bromide,SYBR Green I or Pico Green, the increased density of fluorescence isobserved as the product is increased. By monitoring it, it is possibleto trace the real-time synthetic reaction in a closed system.Application of this type of detection system to the PCR method is alsoconsidered, but it is deemed that there are many problems because thesignal from the product cannot be distinguished from signals from primerdimers etc. However, when this system is applied to this invention, thepossibility of increasing unspecific base pairing is very low, and thushigh sensitivity and low noises can be simultaneously expected. Similarto use of the double-stranded chain-specific intercalater, the transferof fluorescent energy can be utilized for a method of realizing adetection system in a uniform system.

[0118] The method of synthesizing nucleic acid according to the presentinvention is supported by the DNA polymerase catalyzing the stranddisplacement-type reaction for synthesis of complementary chain. Duringthe reaction described above, a reaction step not necessarily requiringthe strand displacement-type polymerase is also contained. However, forsimplification of a constitutional reagent and in an economicalviewpoint, it is advantageous to use one kind of DNA polymerase. As thiskind of DNA polymerase, the following enzymes are known. Further,various mutants of these enzymes can be utilized in the presentinvention insofar as they have both the sequence-dependent activity forsynthesis of complementary chain and the strand displacement activity.The mutants referred to herein include those having only a structurebringing about the catalytic activity required of the enzyme or thosewith modifications to catalytic activity, stability or thermostabilityby e.g. mutations in amino acids.

[0119] Bst DNA polymerase

[0120] Bca (exo-)DNA polymerase

[0121] DNA polymerase I Klenow fragment

[0122] Vent DNA polymerase

[0123] Vent (exo-)DNA polymerase (Vent DNA polymerase deficient inexonuclease activity)

[0124] Deep Vent DNA polymerase

[0125] Deep Vent(exo-)DNA polymerase (Deep Vent DNA polymerase deficientin exonuclease activity)

[0126] Φ29 phage DNA polymerase

[0127] MS-2 phage DNA polymerase

[0128] Z-Taq DNA polymerase (Takara Shuzo Co., Ltd.)

[0129] KOD DNA polymerase (Toyobo Co., Ltd.)

[0130] Among these enzymes, Bst DNA polymerase and Bca (exo-) DNApolymerase are particularly desired enzymes because they have a certaindegree of thermostability and high catalytic activity. The reaction ofthis invention can be carried isothermally in was preferred embodiment,but because of the adjustment of melting temperature (Tm) etc., it isnot always possible to utilize temperature conditions desired for thestability of the enzyme. Accordingly, it is one of the desiredconditions that the enzyme is thermostable. Although the isothermalreaction is feasible, heat denaturation may be conducted to providenucleic acid as a first template, and in this respect too, utilizationof a thermostable enzyme broadens selection of assay protocol.

[0131] Vent (exo-) DNA polymerase is an enzyme having both stranddisplacement activity and a high degree of thermostability. It is knownthat the complementary chain synthetic reaction involving stranddisplacement by DNA polymerase is promoted by adding a single strandbinding protein (Paul M. Lizardi et al., Nature Genetics, 19, 225-232,July, 1998). This action is applied to the present invention, and byadding the single strand binding protein, the effect of promoting thesynthesis of complementary chain can be expected. For example, T4 gene32 is effective as a single strand binding protein for Vent (exo-) DNApolymerase.

[0132] For DNA polymerase free of 3′-5′ exonuclease activity, there is aknown phenomenon where the synthesis of complementary chain does notstop at the 5′-terminal of a template, resulting in generation of aone-base protrusion. In the present invention, this phenomenon is notpreferable because when synthesis of the complementary chain reaches theterminal, the sequence of the 3′-terminal leads to initiation of nextsynthesis of complementary chain. However, because a base “A” is addedat high probability to the 3′-terminal by the DNA polymerase, thesequence may be selected such that the synthesis from the 3′-terminalstarts at “A”, so that there is no problem if an additional base isadded erroneously by DATP. Further, even if the 3′-terminal is protrudedduring synthesis of complementary chain, the 3′→5′ exonuclease activitycan be utilized for digesting the protrusion to make it blunt-ended. Forexample, since natural Vent DNA polymerase has this activity, thisenzyme may be used as a mixture with Vent (exo-) DNA polymerase in orderto solve this problem.

[0133] Various reagents necessary for the method of synthesizing oramplifying nucleic acid according to the present invention may bepreviously packaged and provided as a kit. Specifically, a kit isprovided for the present invention, comprising various kinds ofoligonucleotides necessary as primers for synthesis of complementarychain and as outer primers for displacement, dNTP as a substrate forsynthesis of complementary chain, a DNA polymerase for carrying out thestrand displacement-type synthesis of complementary chain, a buffergiving suitable conditions to the enzyme reaction, and as necessaryregents necessary for detection of synthetic reaction products. Inparticular, the addition of reagents is necessary during the reaction ina preferable mode of the present invention, and thus the reagentsnecessary for one reaction are supplied after pipetted into reactionvessel, whereby the reaction can be initiated by adding only a sample.By constituting a system in which the reaction product can be detectedin situ in a reaction vessel by utilizing a luminescent signal or afluorescent signal, it is not necessary to open and shut the vesselafter reaction. This is very desirable for prevention of contamination.

[0134] The nucleic acid having complementary nucleotide sequencesalternately linked in a single-stranded chain, synthesized according tothe present invention, has e.g. the following usefulness. The firstfeature is use of an advantage resulting from the special structurehaving complementary sequences in one molecule. This feature can beexpected to facilitate detection. That is, there is a known system fordetecting nucleic acid wherein its signal is varied depending on basepairing with a complementary nucleotide sequence. For example, bycombination with the method of utilizing a double-strandedchain-specific intercalater as a detector as described above, adetection system making full use of the characteristics of the syntheticproduct of the present invention can be realized. If the syntheticreaction product of the present invention is once heat-denatured in saiddetection system and returned to the original temperature,intramolecular annealing occurs preferentially thus permittingcomplementary sequences to be rapidly base-paired. If there areunspecific reaction products, they have not complementary sequences inthe molecule so that after separated by heat denaturation into 2 or moremolecules, they cannot immediately be returned to the originaldouble-stranded chain. By providing the step of heat denaturation beforedetection, noises accompanying the unspecific reaction can be reduced.If the DNA polymerase not resistant to heat is used, the step of heatdenaturation has the meaning of termination of the reaction and is thusadvantageous for the control of reaction temperature.

[0135] The second feature is to always form a loop capable of basepairing. The structure of a loop capable of base pairing is shown inFIG. 4. As can be seen from FIG. 4, the loop is composed of thenucleotide sequence F2c (X2c) which can be annealed by the primer and anucleotide sequence intervening between F2c-F1c (X1c). The sequencebetween F2c-F1c (or between X2c-X1c in a more universal form) is anucleotide derived sequence derived from the template. Accordingly, if aprobe having a complementary nucleotide sequence is hybridized with thisregion, template-specific detection is feasible. In addition, thisregion is always ready for base pairing, and therefore, heatdenaturation prior to hybridization is not necessary. The nucleotidesequence constituting a loop in the amplification reaction product inthe present invention may have an arbitrary length. Accordingly, ifhybridization with a probe is desired, a region to be annealed by theprimer and a region to be hybridized by the probe are arrangedseparately to prevent their competition, whereby ideal reactionconditions can be constituted.

[0136] According to a preferable mode of the present invention, a largenumber of loops capable of base pairing are given in a single strand ofnucleic acid. This means that a large number of probes can be hybridizedwith one molecule of nucleic acid to permit highly sensitive detection.It is thus possible to realize not only the improvement of sensitivitybut also a method of detecting nucleic acid based on a special reactionprinciple such as aggregation. For example, a probe immobilized ontofine particles such as polystyrene latex is added to the reactionproduct of the present invention, the aggregation of latex particles isobserved as the hybridization of the product with the probe proceeds.Highly sensitive and quantitative observation is feasible by opticallymeasuring the strength of the aggregation. Because the aggregation canalso be observed with the naked eyes, a reaction system not using anoptical measuring device can also be constituted.

[0137] Further, the reaction product of the present invention permittingmany labels to be bound thereto per nucleic acid molecule enableschromatographic detection. In the field of immunoassay, an analyticalmethod (immunochromatography) using a chromatographic medium utilizing avisually detectable label is used practically. This method is based onthe principle that an analyte is sandwiched between an antibodyimmobilized on a chromatographic medium and a labeled antibody, and theunreacted labeled component is washed away. The reaction product of thepresent invention makes this principle applicable to analysis of nucleicacid. That is, a labeled probe toward the part of a loop is prepared andimmobilized onto a chromatographic medium to prepare a capturing probefor trapping thereby permitting analysis in the chromatographic medium.As the capturing probe, a sequence complementary to the part of the loopcan be utilized. Since the reaction product of the present invention hasa large number of loops, the product binds to a large number of labeledprobes to give a visually recognizable signal.

[0138] The reaction product according to the present invention alwaysgiving a region as a loop capable of base pairing enables a wide varietyof other detection systems. For example, a detection system utilizingsurface plasmon resonance using an immobilized probe for this loopportion is feasible. Further, if a probe for the loop portion is labeledwith a double-stranded chain-specific intercalater, more sensitivefluorescent analysis can be conducted. Alternatively, it is alsopossible to positively utilize the ability of the nucleic acidsynthesized by the present invention to form a loop capable of basepairing at both the 3′- and 5′-sides. For example, one loop is designedto have a common nucleotide sequence between a normal type and anabnormal type, while the other loop is designed to generate a differencetherebetween. It is possible to constitute a characteristic analyticsystem in which the presence of a gene is confirmed by the probe for thecommon portion while the presence of an abnormality is confirmed in theother region. Because the reaction of synthesizing nucleic acidaccording to the present invention can also proceed isothermally, it isa very important advantage that real-time analysis can be effected by ageneral fluorescent photometer. Heretofore, the structure of nucleicacid to be annealed in the same chain is known. However, the nucleicacid having complementary nucleotide sequences linked alternately in asingle-stranded chain obtained by the present invention is novel in thatit contains a large number of loops capable of base pairing with otheroligonucleotides.

[0139] On the other hand, a large number of loops themselves given bythe reaction product according to the present invention can be used asprobes. For, example, in a DNA chip, probes should be accumulated athigh density in a limited area. In present technology, however, thenumber of oligonucleotides which can be immobilized in a certain area islimited. Hence, by use of the reaction product of the present invention,a large number of probes capable of annealing can be immobilized at highdensity. That is, the reaction product according to the presentinvention maybe immobilized as probes on a DNA chip. Afteramplification, the reaction product may be immobilized by any techniquesknown in the art, or the immobilized oligonucleotide is utilized as theoligonucleotide in the amplification reaction of the present invention,resulting in generating the immobilized reaction product. By use of theprobe thus immobilized, a large number of sample DNAs can be hybridizedin a limited area, and as a result, high signals can be expected.

BRIEF DESCRIPTION OF THE DRAWINGS

[0140]FIG. 1 is an illustration of a part (1) to (4) of the reactionprinciple in a preferable mode of the present invention.

[0141]FIG. 2 is an illustration of a part (5) to (7) of the reactionprinciple in a preferable mode of the present invention.

[0142]FIG. 3 is an illustration of a part (8) to (10) of the reactionprinciple in a preferable mode of the present invention.

[0143]FIG. 4 is an illustration of the structure of a loop formed by thesingle-stranded nucleic acid according to the present invention.

[0144]FIG. 5 is an illustration of a part (A) to (B) in a basic mode ofthe present invention.

[0145]FIG. 6 is an illustration of a part (C) to (D) in a basic mode ofthe present invention.

[0146]FIG. 7 is a drawing showing the positional relationship of eachnucleotide sequence constituting an oligonucleotide in the targetnucleotide sequence of M13mp18.

[0147]FIG. 8 is a photograph showing the result of agaroseelectrophoresis of a product obtained by the method of synthesizingsingle-stranded nucleic acid with M13mp18 as a template according to thepresent invention.

[0148] Lane 1: XIV size marker

[0149] Lane 2: 1 fmol M13mp18 dsDNA

[0150] Lane 3: No target

[0151]FIG. 9 is a photograph showing the result of agarose gelelectrophoresis of a restriction enzyme-digested product obtained inExample 1 by the nucleic acid synthetic reaction according to thepresent invention.

[0152] Lane 1: XIV size marker

[0153] Lane 2: BamHI digest of the purified product

[0154] Lane 3: PvuII digest of the purified product

[0155] Lane 4: HindIII digest of the purified product

[0156]FIG. 10 is a photograph showing the result of agarose gelelectrophoresis of a product obtained by the method of synthesizingsingle-stranded nucleic acid according to the present invention usingM13mp18 as a template in the presence of betaine. 0, 0.5, 1 and 2indicate the concentration (M) of betaine added to the reactionsolution. N indicates the negative control, and -21 indicates theconcentration 10⁻²¹ mol of template DNA.

[0157]FIG. 11 is a drawing showing the positional relationship of eachnucleotide sequence constituting an oligonucleotide in a targetnucleotide sequence derived from HVB.

[0158]FIG. 12 is a photograph showing the result of agarose gelelectrophoresis of a product obtained by the method of synthesizingsingle-stranded nucleic acid according to the present invention whereinHBV-M13mp18 integrated in M13mp18 was used as a template.

[0159] Lane 1: XIV size marker

[0160] Lane 2: 1 fmol HBV-M13mp18 dsDNA

[0161] Lane 3: No target

[0162]FIG. 13 is a photograph showing the result of gel electrophoresisof an alkali-denatured product obtained by the method of synthesizingsingle-stranded nucleic acid according to the present invention.

[0163] Lane 1: HindIII-digested fragment from lambda-phage

[0164] Lane 2: The reaction product in Example 1.

[0165] Lane 3: The reaction product in Example 3.

[0166]FIG. 14 is a photograph showing the result of agarose gelelectrophoresis of a product obtained by the method of synthesizingsingle-stranded nucleic acid according to the present invention whereinthe concentration of M13mp18 as a target was varied. The upper and lowerphotographs show the result of the reaction for 1 and 3 hoursrespectively.

[0167] Lane 1: M13mp18 dsDNA 1×10⁻¹⁵ mol/tube

[0168] Lane 2: M13mp18 dsDNA 1×10⁻¹⁶ mol/tube

[0169] Lane 3: M13mp18 dsDNA 1×10⁻¹⁷ mol/tube

[0170] Lane 4: M13mp18 dsDNA 1×10⁻¹⁸ mol/tube

[0171] Lane 5: M13mp18 dsDNA 1×10⁻¹⁹ mol/tube

[0172] Lane 6: M13mp18 dsDNA 1×10⁻²⁰ mol/tube

[0173] Lane 7: M13mp18 dsDNA 1×10⁻²¹ mol/tube

[0174] Lane 8: M13mp18 dsDNA 1×10⁻²² mol/tube

[0175] Lane 9: No target

[0176] Lane 10: XIV size marker

[0177]FIG. 15 is a drawing showing the position of a mutation and thepositional relationship of each region toward a target nucleotidesequence (target). Underlined guanine is replaced by adenine in themutant.

[0178]FIG. 16 is a photograph showing the result of agarose gelelectrophoresis of a product according to the amplification reaction ofthe present invention.

[0179] M: 100 bp ladder (New. England Biolabs)

[0180] N: No template (purified water)

[0181] WT: 1 fmol wild-type template M13mp18

[0182] MT: 1 fmol mutant template M13mp18FM

[0183]FIG. 17, is a drawing showing the positional relationship of eachnucleotide sequence constituting an oligonucleotide in a nucleotidesequence coding for target mRNA.

[0184]FIG. 18 is a photograph showing the result of agaroseelectrophoresis of a product obtained by the method of synthesizingsingle-stranded nucleic acid according to the present invention usingmRNA as a target.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLE 1 Amplification of aRegion in M13mp18

[0185] The method of synthesizing the nucleic acid having complementarychains alternately linked in a single-stranded chain according to thepresent invention was attempted using M13mp18 as a template. Four kindsof primers, that is, M13FA, M13RA, M13F3, and M13R3, were used in theexperiment. M13F3 and M13R3 were outer primers for displacing the firstnucleic acid obtained respectively with M13FA and M13RA as the origin ofsynthesis. Because the outer primers are primers serving as the originof synthesis of complementary chain after synthesis with M13FA (orM13RA), these were designed to anneal to a region contiguous to M13FA(or M13RA) by use of the phenomenon of contiguous stacking. Further, theconcentrations of these primers were set high such that annealing ofM13FA (or M13RA) occurred preferentially.

[0186] The nucleotide sequence constituting each primer is as shown inthe Sequence Listing. The structural characteristics of the primers aresummarized below. Further, the positional relationship of each regiontoward the target nucleotide sequence (target) is shown in FIG. 7.

[0187] Primer Region at the 5′-side/region at the 3′-side

[0188] M13FA The same as region F1c in complementary chain synthesizedby M13FA/complementary to region F2c in M13mp18

[0189] M13RA The same as region R1c in complementary chain synthesizedby M13RA/complementary to region R2c in complementary chain synthesizedby M13FA

[0190] M13F3 Complementary to F3c contiguous to the 3′-side of regionF2c in M13mp18

[0191] M13R3 Complementary to R3c contiguous to the 3′-side of regionF2c in complementary chain synthesized by M13FA

[0192] By such primers, nucleic acid wherein a region extending from F1cto R1c in M13mp18, and its complementary nucleotide sequence, arealternately linked via a loop-forming sequence containing F2c in asingle-stranded chain, is synthesized. The composition of a reactionsolution for the method of synthesizing nucleic acid by these primersaccording to the present invention is shown below.

[0193] Composition of the reaction solution (in 25 μL)

[0194] 20 mM Tris-HCl pH8.8

[0195] 10 mM KCl

[0196] 10 mM (NH₄)₂SO₄

[0197] 6 mm MgSO₄

[0198] 0.1% Triton X-100

[0199] 5% dimethyl sulfoxide (DMSO)

[0200] 0.4 mM dNTP

[0201] Primers:

[0202] 800 nm M13FA/SEQ ID NO:1

[0203] 800 nm M13RA/SEQ ID NO:2

[0204] 200 nm M13F3/SEQ ID NO:3

[0205] 200 nm M13R3/SEQ ID NO:4

[0206] Target: M13mp18 dsDNA/SEQ ID NO:5

[0207] Reaction: The above reaction solution was heated at 95° C. for 5minutes, and the target was denatured into a single-stranded chain. Thereaction solution was transferred to ice-cold water, and 4 U of Bst DNApolymerase (NEW ENGLAND Biolabs) was added thereto and the mixture wasreacted at 65° C. 1 hour. After reaction, the reaction was terminated at80° C. for 10 minutes and transferred again to ice-cold water.

[0208] Confirmation of the reaction: 1 μl loading buffer was added to 5μl of the above reaction solution, and the sample was ectrophoresed for1 hour at 80 mV on 2% agarose gel (0.5% TBE). As a molecular-weightmarker, XIV (100 bp ladder, Boehringer Mannheim) was used. The gel afterelectrophoresis was stained with SYBR Green I (Molecular probes, Inc.)to confirm the nucleic acid. The results are shown in FIG. 8. Therespective lanes correspond to the following samples.

[0209] 1. XIV size marker.

[0210] 2. 1 fmol M13mp18 dsDNA.

[0211] 3. No target.

[0212] In lane 3, no band was confirmed except that the unreactedprimers were stained. In lane 2 in the presence of the target, theproducts were confirmed as a low size band ladder, as smeared stainingat high size and as a band hardly electrophoresed in the gel. Among thelow-size bands, bands in the vicinity of 290 bp and 450 bp agree withthe products estimated in the synthetic reaction of this invention, thatis, double-stranded chains of SEQ ID NOS: 11 and 12 (corresponding todouble-stranded chains formed as shown in FIGS. 2-(7) and 2-(10)) and asingle-stranded chain of SEQ ID NO: 13 (corresponding to the longsingle-stranded chain in FIG. 3-(9)), and it was thus confirmed that thereaction proceeds as expected. It was estimated that the electrophoresisresults of the smeared pattern at high size and the band notelectrophoresed were brought about because this reaction was basically acontinuous reaction to permit varying molecular weights of the reactionproduct and further because the product has, a complicated structurehaving a partially single-stranded chain and a double-stranded complex.

EXAMPLE 2 Confirmation of the Reaction Products by Digestion withRestriction Enzymes

[0213] For the purpose of clarifying the structure of the nucleic acidhaving complementary nucleotide sequences linked alternately in asingle-stranded chain obtained in Example 1 according to the presentinvention, the digestion of the products with restriction enzymes wasconducted. If fragments are theoretically generated by digestion thereofwith restriction enzymes and simultaneously the smear pattern at highsize and the band not electrophoresed as observed in Example 1disappear, then it can be estimated that any of these products are thenucleic acid having complementary sequences linked alternately in asingle-stranded chain synthesized according to the present invention.

[0214] The reaction solution (200 μl ) from 8 tubes in Example 1 waspooled and purified by treatment with phenol and precipitation withethanol. The resulting precipitates were recovered and dissolved againin 200 μl TE buffer, and 10 μl aliquot was digested at 37° C. for 2hours with restriction enzymes BamHI, PvuII, and HindIII respectively.The digest was electrophoresed for 1 hour at 80 mV on 2 agarose gel(0.5% TBE). As a molecular marker, Super Ladder-Low (100 bp ladder)(Gensura Laboratories, Inc.) was used. The gel after electrophoresis wasstained with SYBR Green I (Molecular Probes, Inc.) to confirm thenucleic acid. The results are shown in FIG. 9. The respective lanescorrespond to the following samples.

[0215] 1. XIV size marker

[0216] 2. BamHI digest of the purified product.

[0217] 3. PvuII digest of the purified product

[0218] 4. HindIII digest of the purified product

[0219] It is estimated that nucleotide sequences constituting relativelyshort amplification products are those of SEQ ID NOS:13, 14, 15 and 16.From these nucleotide sequences, the estimated size of each fragmentdigested with the restriction enzymes is as shown in Table 1. “L” in thetable indicates that its position in electrophoresis is not establishedbecause L is a fragment containing a loop (single stranded chain). TABLE1 Restriction enzyme-digested fragments of the amplification productsaccording to the present invention SEQ ID NO BamHI PvuII HindIII 13177 + L  56 + L 147 + L 14  15 + 101 + L — 142 + L 15 171 + 101 + L 56 + L 147 + 161 + L 16  11 + 101 + 230 + L 237 + L 142 + 170 + LSummary 101, 177, 230 56, 237 142, 147, 161, 170

[0220] Because almost all bands before digestion were changed intoestimated bands, it was confirmed that the object reaction products wereamplified. Further, it was also shown that there were no or lessunspecific products.

EXAMPLE 3 Promotion of Amplification Reaction by Addition of Betaine

[0221] An experiment for examining the effect of betaine(N,N,N-trimethylglycine, Sigma) added to the amplification reactionsolution on the amplification reaction of nucleic acid was conducted.Synthesis of the nucleic acid having complementary chains alternatelylinked in a single-stranded chain according to the present invention wasconducted using M13mp18 as a template similarly in Example 1 in thepresence of betaine at various concentrations. The primers used in theexperiment were identical to those used in Example 1. The amount of thetemplate DNA was 10⁻²¹ mol (M13mp18) and water was used as the negativecontrol. Betaine was added at concentrations of 0, 0.5, 1 and 2 M to thereaction solution. The composition of the reaction solution is shownbelow.

[0222] Composition of the reaction solution (in 25 μL)

[0223] 20 Tris-HCl pH8.8

[0224] 4 mM MgSO₄

[0225] 0.4 mM dNTPs

[0226] 10 mM KCl

[0227] 10 mM (NH₄)₂SO₄

[0228] 0.1% Triton X-100

[0229] Primers:

[0230] 800 nM M13FA/SEQ ID NO:1

[0231] 800 nM M13RA/SEQ ID NO:2

[0232] 200 nM M13F3/SEQ ID NO:3

[0233] 200 nM M13R3/SEQ ID NO:4

[0234] Target: M13mp18 dsDNA/SEQ ID NO:5

[0235] The polymerase, reaction conditions, and conditions forelectrophoresis after the reaction were identical to those described inExample 1.

[0236] The results are shown in FIG. 10. In the reaction in the presenceof betaine at a concentration of 0.5 or 1.0 M, the amount of theamplification product was increased. Further, if its concentration wasincreased to 2.0 M, no amplification was observed. It was thus shownthat the amplification reaction was promoted in the presence of betaineat a suitable concentration. The estimated reason that the amount of theamplification product was decreased when the concentration of betainewas 2 M was that Tm was lowered too much.

EXAMPLE 4 Amplification of HBV Gene Sequence

[0237] The method of synthesizing nucleic acid according to the presentinvention was attempted wherein M13mp18 dsDNA having a partial sequenceof HBV gene integrated therein was used as a template. Four kinds ofprimers, HB65FA (SEQ ID NO:6), HB65RA (SEQ ID NO:7), HBF3 (SEQ ID NO:8)and HBR3 (SEQ ID NO:9), were used in the experiment. HBF3 and HBR3 wereouter primers for displacement of the first nucleic acid obtainedrespectively with HB65FA and HB65RA as the origin of synthesis. Becausethe outer primers are primers serving as the origin of synthesis ofcomplementary chain after synthesis with HB65FA (or HB65RA), these weredesigned to anneal to a region contiguous to HB65FA (or HB65RA) by useof the phenomenon of contiguous stacking. Further, the concentrations ofthese primers were set high such that annealing of HB65FA (or HB65RA)occurred preferentially. The target sequence (430 bp) in this example,derived from HBV integrated in M13mp18, is shown in SEQ ID NO:10.

[0238] The nucleotide sequence constituting each primer is shown in theSequence Listing. The structural feature of each primer is summarizedbelow. Further, the positional relationship of each region toward thetarget nucleotide sequence (target) is shown in FIG. 11.

[0239] Primer Region at the 5′-side/region at the 3′-side

[0240] HB65FA The same as region F1c in complementary chain synthesizedby HB65FA/complementary to region F2c in HBV-M13mp18

[0241] HB65RA The same as region R1c in complementary chain synthesizedby HB65RA/complementary to region R2c in complementary chain synthesizedby HB65FA

[0242] HBF3 Complementary to F3c contiguous to the 3′-side of region F2cin HBV-M13mp18

[0243] HBR3 Complementary to R3c contiguous to the 3′-side of region F2cin complementary chain synthesized by HB65FA

[0244] By such primers, nucleic acid wherein a region extending from F1cto R1c in M13mp18 (HBV-M13mp18) having a partial sequence of HBV geneintegrated therein, and its complementary nucleotide sequence, arealternately linked via a loop-forming sequence containing F2c in asingle-stranded chain, is synthesized. The reaction was conducted underthe same conditions as in Example 1 except that the primers describedabove were used, and the reaction solution was analyzed by agaroseelectrophoresis. The results are shown in FIG. 12. The respective lanescorrespond to the following samples.

[0245] 1. XIV size marker

[0246] 2. 1 fmol HBV-M13mp18 dsDNA.

[0247] 3. No target

[0248] Similar to Example 1, the products were confirmed only in thepresence of the target as a low size band ladder, as smeared staining athigh size and as a band hardly electrophoresed in the gel (lane 2).Among the low-size bands, bands in the vicinity of 310 bp and 480 bpagree with the products estimated in the synthetic reaction of thisinvention, that is, double-stranded chains of SEQ ID NOS:17 and 18, andit was thus confirmed that the reaction proceeds as expected. Asdescribed in the results in Example 1, it was estimated that the smearedpattern at high size and the band not electrophoresed were caused by thestructure of the synthetic product characteristic of the presentinvention. From this experiment, it was confirmed that the presentinvention can be practiced even if a different sequence (target) is usedfor amplification.

EXAMPLE 5 Confirmation of the Sizes of the Synthetic Reaction Products

[0249] To confirm the structure of the nucleic acid synthesizedaccording to the present invention, its length was analyzed byelectrophoresis under alkali-denaturing conditions. 1 μl alkalineloading buffer was added to 5 μl of each reaction solution in thepresence of the target in Example 1 or 4 and electrophoresed at 50 mA in0.7% agarose gel (50 mM NaOH, 1 mm EDTA) for 14 hours. As themolecular-weight size marker, HindIII-digested lambda-phage fragmentswere used. The gel after electrophoresis was neutralized with 1 M Tris,pH 8 and stained with SYBR Green I (Molecular Probes, Inc.) to confirmthe nucleic acid. The results are shown in FIG. 13. The respective lanescorrespond to the following samples.

[0250] 1. HindIII-digested fragments from lambda-phage.

[0251] 2. The reaction product in Example 1.

[0252] 3. The reaction product in Example 4.

[0253] When the reaction product was electrophoresed underalkali-denaturing conditions, its size in a single-stranded state couldbe confirmed. It was confirmed that the sizes of the major products inboth Example 1 (lane 2) and Example 4 (lane 3) were within 2 kbase.Further, it was revealed that the product according to the presentinvention had been extended to have a size of at least 6 kbase or morewithin the range capable of confirmation by this analysis. In addition,it was confirmed again that bands not electrophoresed undernon-denaturing conditions in Examples 1 and 4 were separated in adenatured state into individual single-stranded chains of smaller size.

EXAMPLE 6 Confirmation of Amplification Depending on the Concentrationof a Target in the Amplification of a Region in M-13mp13

[0254] The influence of a varying concentration of a target on themethod of synthesizing nucleic acid according to the present inventionwas observed. The method of synthesizing nucleic acid according to thepresent invention was carried out under the same conditions as inExample 1 except that the amount of M13mp18 dsDNA as the target was 0 to1 fmol and the reaction time was 1 hour or 3 hours. Similar to Example1, the sample was electrophoresed in 2% agarose gel (0.5% TBE) andstained with SYBR Green I (Molecular Probes, Inc.) to confirm thenucleic acid. As a molecular size marker, XIV (100 bp ladder, BoehringerMannheim) was used. The results are shown in FIG. 14 (upper: 1-hourreaction, below: 3-hour reaction). The respective lanes correspond tothe following samples:

[0255] 1. M13mp18 dsDNA 1×10⁻¹⁵ mol/tube.

[0256] 2. M13mp18 dsDNA 1×10⁻¹⁶ mol/tube.

[0257] 3. M13mp18 dsDNA 1×10⁻¹⁷ mol/tube.

[0258] 4. M13mp18 dsDNA 1×10⁻¹⁸ mol/tube.

[0259] 5. M13mp18 dsDNA 1×10⁻¹⁹ mol/tube.

[0260] 6. M13mp18 dsDNA 1×10⁻²⁰ mol/tube.

[0261] 7. M13mp18 dsDNA 1×10⁻²¹ mol/tube.

[0262] 8. M13mp18 dsDNA 1×10⁻²² mol/tube.

[0263] 9. No target.

[0264] 10. XIV size marker.

[0265] A common band among the respective lanes appears in a lower partin the electrophoretic profile and shows the unreacted stained primers.Regardless of the reaction time, no amplification product is observed inthe absence of the target. A staining pattern, depending on theconcentration of the target, of the amplification product was obtainedonly in the presence of the target. Further, the amplification productcould be confirmed at lower concentration as the reaction time wasincreased.

EXAMPLE 7 Detection of a Point Mutation

[0266] (1) Preparation of M13mp18FM (Mutant)

[0267] The target DNA used was M13mp18 (wild-type) and M13mp18FM(mutant). For the construction of the mutant M13mp18FM, LA PCR™ in vitroMutagenesis Kit (Takara Shuzo Co., Ltd.) was used to replace onenucleotide for mutation. Thereafter, the sequence was confirmed bysequencing. The sequence of the F1 region is shown below: Wild-type:CCGGGGATCCTCTAGAGTCG (SEQ ID NO:19) Mutant: CCGGGGATCCTCTAGAGTCA (SEQ IDNO:20)

[0268] (2) Design of Primers

[0269] The FA primers used for the wild-type and the mutant wereprovided at the 5′-terminal of the F1c region thereof with differentnucleotide sequences, respectively. The location of the mutation and thepositional relationship of each region toward the target nucleotidesequence (target) are shown in FIG. 15.

[0270] (3) Amplification Reaction

[0271] An experiment was conducted to examine whether template-specificamplification reaction occurs using a combination of specific primersshown below by use of M13mp18 (wild-type) and M13mp18FM (mutant) asprimers.

[0272] Primer set for wild-type amplification: FAd4, RAd4, F3, R3

[0273] Primer set for mutant amplification: FAMd4, RAd4, F3, R3

[0274] The nucleotide sequence of each primer is as follows: FAd4:CGACTCTAGAGGATCCCCGGTTTTTGTTGTGTGGAATTGTGAGCGGAT (SEQ ID NO:21) FAMd4:TGACTCTAGAGGATCCCCGGTTTTTGTTGTGTGGAATTGTGAGCGGAT (SEQ ID NO:22) RAd4:CGTCGTGACTGGGAAAACCCTTTTTGTGCGGGCCTCTTCGCTATTAC (SEQ ID NO:23) F3:ACTTTATGCTTCCGGCTCGTA (SEQ ID NO:24) R3: GTTGGGAAGGGCGATCG (SEQ IDNO:25)

[0275] (4) Detection of the Point Mutation in M13mp18

[0276] The composition of the reaction solution is as follows: Finalconcentration D2W 3.75 μL 10 × Thermo pol buffer(NEB)  2.5 μL  20 mMTris-HCl pH 8.8  10 mM KCl  10 mM (NH₄)₂SO₄  6 mM MgSO₄ 0.1% TritonX-100 2.5 mM dNTP   4 μL 400 μM  100 mM MgSO₄  0.5 μL   4 M Betaine 6.25 μL 1 M M13FAd4 primer (10 pmol/μL) or   2 μL 800 nM M13FAMd4 primer (10pmol/μL) M13RAd4 primer (10 pmol/μL)   2 μL 800 nM M13F3 primer (10pmol/μL)  0.5 μL 200 nM M13R3 primer (10 pmol/μL)  0.5 μL 200 nM Totalamount   22 μL

[0277] 1 fmol (2 μl) of the target M13mp18 or M13mp18FM was added to thereaction solution and heated at 95° C. for 5 minutes whereby the targetwas denatured into a single-stranded chain. The reaction solution wastransferred to ice-cold water, and 1 μl (8 U) of Bst DNA polymerase (NEWENGLAND Biolab) was added thereto and reacted for 1 hour at 68° C.or68.5° C. After reaction, the reaction was terminated at 80° C. for 10minutes, and the reaction solution was transferred again to ice-coldwater.

[0278] As shown in FIG. 16, when FAd4 for wild type was used as the FAprimer, effective amplification was observed only in the presence of thewild-type template. On the other hand, when FAMd4 for mutant was used asthe FA primer, effective amplification was observed only in the presenceof the wild-type [sic.] template.

[0279] From the results described above, it was shown that the pointmutation could be detected efficiently by use of the amplificationreaction of the present invention.

EXAMPLE 8 Amplification Reaction of mRNA as a Target

[0280] The method of synthesizing nucleic acid according to the presentinvention was attempted using mRNA as the target nucleic acid. Toprepare the target mRNA, prostate cancer cell line LNCaP cells (ATCC No.CRL-1740) expressing prostate specific antigen (PSA) were mixed withchronic myeloid leukemia cell line K562 cells (ATCC No. CCL-243) asnon-expressing cells at 1: 10⁶ to 100: 10⁶, followed by extraction ofthe total RNA by use of an RNeasy Mini kit from Qiagen (Germany). Fourkinds of primers, that is, PSAFA, PSARA, PSAF3 and PSAR3, were used inthe experiment. PSAF3 and PSAR3 are outer primers for displacing thefirst nucleic acid obtained respectively with PSAFA and PSARA as theorigin of synthesis. Further, the concentrations of these primers wereset high such that annealing of PSAFA (or PSARA) occurredpreferentially. The nucleotide sequences constituting the respectiveprimers are as follows.

[0281] Primer: PSAFA: TGTTCCTGATGCAGTGGGCAGCTTTAGTCTGCGGCGGTGTTCTG (SEQID NO:26) PSARA: TGCTGGGTCGGCACAGCCTGAAGCTGACCTGAAATACCTGGCCTG (SEQ IDNO:27) PSAF3: TGCTTGTGGCCTCTCGTG (SEQ ID NO:28) PSAR3: GGGTGTGGGAAGCTGTG(SEQ ID NO:29)

[0282] The structural features of the primers are summarized below.Further, the positional relationship of each primer toward the DNAnucleotide sequence coding for the target mRNA and recognition sites ofrestriction enzyme Sau3AI are shown in FIG. 17.

[0283] Primer Region at the 5′-side/region at the 3′-side

[0284] PSAFA The same as region F1c in complementary chain synthesizedby PSAFA/complementary to region F2c in the target nucleotide sequence

[0285] PSARA The, same as region R1c in complementary chain synthesizedby PSARA/complementary to region R2c in complementary chain synthesizedby PSAFA

[0286] PSAF3 Complementary to F3c contiguous to the 3′-side of regionF2c in the target nucleotide sequence

[0287] PSAR3. Complementary to R3c contiguous to the 3′-side of regionR2c in complementary chain synthesized by PSAFA

[0288] The composition of a reaction solution for the method ofsynthesizing nucleic acid according to the present invention is asfollows:

[0289] Composition of the reaction solution (in 25 μL)

[0290] 20 mM Tris-HCl pH 8.8

[0291] 4 mM MgSO₄

[0292] 0.4 mM dNTPs

[0293] 10 mM KCl

[0294] 10 mM (NH₄)₂SO₄

[0295] 0.1% Triton X-100

[0296] 0.8 M betaine

[0297] 5 mM DTT

[0298] 1600 nM PSAFA & PSARA primer

[0299] 200 nM PSAF3 & PSAR3 primer

[0300] 8 U Bst DNA polymerase

[0301] 100 U Rever Tra Ace (Toyobo Co., Ltd., Japan)

[0302] 5 μg total RNA

[0303] All ingredients were mixed on ice. In this experiment, mRNA(single-stranded chain) is used as a target, and thus the step of makingsingle-stranded chain by heat denaturation is not necessary. Thereaction was conducted at 65° C. for 45 minutes, and the reaction wasterminated at 85° C. for 5 minutes. After reaction, 5 μl of the reactionsolution was electrophoresed in 2% agarose and detected by SYBR Green I.

[0304] The results are shown in Table 18. The respective lanescorrespond to the following samples. Lane Bst RT Number of LNCaPcells/10⁶ K562 cells 1 − + 0 2 − + 10 3 + − 0 4 + − 10 5 + + 0 6 + + 17 + + 10 8 Sau3AI digest of 1 μL aliquot of the reaction solution inlane 6 9 Sau3AI digest of 1 μL aliquot of the reaction solution in lane7 10 Size maker, 100 bp ladder (New England Biolabs)

[0305] In the absence of either Bst DNA polymerase or Rever Tra Ace, noamplification product could be obtained (lanes 1 to 4). In the presenceof both the enzymes, an amplification product was detected (lanes 5 to7) if RNA derived from LNCaP was present. RNA extracted from one LNCaPcell/one million K562 cells could be detected (lane 6). When theamplification product was digested at the restriction enzyme site Sau3AIlocated in the inside of the target, the product was digested into afragment of estimated size (lanes 8 and 9).

[0306] From the results described above, it was confirmed that thedesired reaction product can be obtained in the method of synthesizingnucleic acid according to the present invention even if RNA is used as atarget.

[0307] Industrial Applicability

[0308] According to the novel oligonucleotide according to the presentinvention and the method of synthesizing nucleic acid by using saidoligonucleotide, there is provided a method of synthesizing nucleic acidhaving complementary nucleotide sequences linked alternately in asingle-stranded chain, without requiring any complicated control oftemperature. A complementary chain synthesized with the oligonucleotideas a primer based on the present invention serves as the origin ofsynthesizing a new complementary chain with the 3′-terminal of saidsynthesized chain as a template. This is accompanied by formation of aloop causing annealing of a new primer, and a product of the reaction ofsynthesizing complementary chain with the previously synthesized chainas a template is displaced again by synthesis of complementary chainfrom the loop and made ready for base pairing. The thus obtained nucleicacid synthesized with itself as a template is combined with e.g. a knownnucleic acid synthesizing method such as SDA, to contribute theimprovement of efficiency of nucleic acid synthesis.

[0309] According to an additional preferred mode of the presentinvention, there is provided a novel method of synthesizing nucleicacid, which achieves the improvement of efficiency of the known methodof synthesizing nucleic acid, does not require complicated control oftemperature, can be expected to attain high efficiency of amplificationand can achieve high specificity. That is, the oligonucleotide based onthe present invention is applied to a template chain and itscomplementary chain whereby nucleic acid having complementary sequenceslinked alternately in a single-stranded chain can be successivelysynthesized. This reaction continues theoretically until the startingmaterials necessary for synthesis are exhausted, during which newnucleic acid initiated to be synthesized from the loop continues to beformed. The elongation from the oligonucleotide having annealed to theloop performs strand displacement for supplying 3′-OH for elongation oflong single-stranded nucleic acid (that is, nucleic acid having pluralpairs of complementary chains linked therein). On the other hand, the3′-OH of the long single-stranded chain performs the reaction ofsynthesizing complementary chain with itself as a template whereby itselongation is achieved, during which a new complementary chain whosesynthesis is initiated from the loop is displaced. Such an amplificationreaction step proceeds under isothermal conditions while maintaininghigh specificity.

[0310] The oligonucleotides in the present invention can, when twocontiguous regions are arranged as designed, function as primers for thereaction of synthesizing nucleic acid according to the presentinvention. This contributes significantly to the preservation ofspecificity. By comparison with e.g. PCR where unspecific amplificationreaction is initiated by unspecific missannealing regardless of theintended positional relationship of 2 primers, it can be easilyexplained that high specificity can be expected in the presentinvention. This feature can be utilized to detect SNPs highlysensitively and accurately.

[0311] The characterizing feature of the present invention lies in thatsuch reaction can be easily achieved by a very simple constitution ofreagents. For example, the oligonucleotide according to the presentinvention has a special structure, but this is a matter of selection ofnucleotide sequence, and it is a simple oligonucleotide as substance.Further, in a preferred mode, the reaction can proceed by only a DNApolymerase catalyzing the strand displacement-type reaction ofsynthesizing complementary chain. Further, if the present invention iscarried out with RNA as a template, a DNA polymerase such as Bca DNApolymerase having both reverse transcriptase activity and stranddisplacement-type DNA polymerase activity is used so that all enzymereactions can be conducted by the single enzyme. The reaction principleof realizing a high degree of nucleic acid amplification reaction bysuch simple enzyme reaction is not known. Even for the application ofthe present invention to a known nucleic acid synthesizing reaction suchas SDA, no additional enzyme is necessary for their combination, andsuch a simple combination with the oligonucleotide based on the presentinvention can be applied to various reaction systems. Accordingly, itcan be said that the method of synthesizing nucleic acid according tothe present invention is also advantageous in respect of cost.

[0312] As described above, the method of synthesizing nucleic acidaccording to the present invention and the oligonucleotide thereforprovide a new principle of simultaneously solving a plurality ofdifficult problems such as operativeness (temperature control is notnecessary), improvement of efficiency of synthesis, economization, andhigh specificity.

1 29 1 52 DNA Artificial Sequence Description of Artificial SequenceArtificially synthesized primer sequence 1 cgactctaga ggatccccgggtactttttg ttgtgtggaa ttgtgagcgg at 52 2 51 DNA Artificial SequenceDescription of Artificial Sequence Artificially synthesized primersequence 2 acaacgtcgt gactgggaaa accctttttg tgcgggcctc ttcgctatta c 51 321 DNA Artificial Sequence Description of Artificial SequenceArtificially synthesized primer sequence 3 actttatgct tccggctcgt a 21 417 DNA Artificial Sequence Description of Artificial SequenceArtificially synthesized primer sequence 4 gttgggaagg gcgatcg 17 5 600DNA Bacteriophage M13mp18 5 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccgattcattaat gcagctggca 60 cgacaggttt cccgactgga aagcgggcag tgagcgcaacgcaattaatg tgagttagct 120 cactcattag gcaccccagg ctttacactt tatgcttccggctcgtatgt tgtgtggaat 180 tgtgagcgga taacaatttc acacaggaaa cagctatgaccatgattacg aattcgagct 240 cggtacccgg ggatcctcta gagtcgacct gcaggcatgcaagcttggca ctggccgtcg 300 ttttacaacg tcgtgactgg gaaaaccctg gcgttacccaacttaatcgc cttgcagcac 360 atcccccttt cgccagctgg cgtaatagcg aagaggcccgcaccgatcgc ccttcccaac 420 agttgcgcag cctgaatggc gaatggcgct ttgcctggtttccggcacca gaagcggtgc 480 cggaaagctg gctggagtgc gatcttcctg aggccgatacggtcgtcgtc ccctcaaact 540 ggcagatgca cggttacgat gcgcccatct acaccaacgtaacctatccc attacggtca 600 6 63 DNA Artificial Sequence Description ofArtificial Sequence Artificially synthesized primer sequence 6ctcttccaaa agtaaggcag gaaatgtgaa accagatcgt aatttggaag acccagcatc 60 cag63 7 43 DNA Artificial Sequence Description of Artificial SequenceArtificially synthesized primer sequence 7 gtggattcgc actcctcccgctgatcggga cctgcctcgt cgt 43 8 16 DNA Artificial Sequence Description ofArtificial Sequence Artificially synthesized primer sequence 8gccacctggg tgggaa 16 9 22 DNA Artificial Sequence Description ofArtificial Sequence Artificially synthesized primer sequence 9ggcgagggag ttcttcttct ag 22 10 430 DNA Hepatitis B virus 10 ctccttgacaccgcctctgc tctgtatcgg gaggccttag agtctccgga acattgttca 60 cctcaccatacagcactcag gcaagctatt ctgtgttggg gtgagttaat gaatctggcc 120 acctgggtgggaagtaattt ggaagaccca gcatccaggg aattagtagt cagctatgtc 180 aatgttaatatgggcctaaa aatcagacaa ctattgtggt ttcacatttc ctgccttact 240 tttggaagagaaactgtttt ggagtatttg gtatcttttg gagtgtggat tcgcactcct 300 cccgcttacagaccaccaaa tgcccctatc ttatcaacac ttccggaaac tactgttgtt 360 agacgacgaggcaggtcccc tagaagaaga actccctcgc ctcgcagacg aaggtctcaa 420 tcgccgcgtc430 11 293 DNA Artificial Sequence Description of Artificial SequenceArtificially synthesized sequence 11 acaacgtcgt gactgggaaa accctttttgtgcgggcctc ttcgctatta cgccagctgg 60 cgaaaggggg atgtgctgca aggcgattaagttgggtaac gccagggttt tcccagtcac 120 gacgttgtaa aacgacggcc agtgccaagcttgcatgcct gcaggtcgac tctagaggat 180 ccccgggtac cgagctcgaa ttcgtaatcatggtcatagc tgtttcctgt gtgaaattgt 240 tatccgctca caattccaca caacaaaaagtacccgggga tcctctagag tcg 293 12 293 DNA Artificial Sequence Descriptionof Artificial Sequence Artificially synthesized sequence 12 cgactctagaggatccccgg gtactttttg ttgtgtggaa ttgtgagcgg ataacaattt 60 cacacaggaaacagctatga ccatgattac gaattcgagc tcggtacccg gggatcctct 120 agagtcgacctgcaggcatg caagcttggc actggccgtc gttttacaac gtcgtgactg 180 ggaaaaccctggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 240 gcgtaatagcgaagaggccc gcacaaaaag ggttttccca gtcacgacgt tgt 293 13 459 DNAArtificial Sequence Description of Artificial Sequence Artificiallysynthesized sequence 13 acaacgtcgt gactgggaaa accctttttg tgcgggcctcttcgctatta cgccagctgg 60 cgaaaggggg atgtgctgca aggcgattaa gttgggtaacgccagggttt tcccagtcac 120 gacgttgtaa aacgacggcc agtgccaagc ttgcatgcctgcaggtcgac tctagaggat 180 ccccgggtac cgagctcgaa ttcgtaatca tggtcatagctgtttcctgt gtgaaattgt 240 tatccgctca caattccaca caacaaaaag tacccggggatcctctagag tcgacctgca 300 ggcatgcaag cttggcactg gccgtcgttt tacaacgtcgtgactgggaa aaccctggcg 360 ttacccaact taatcgcctt gcagcacatc cccctttcgccagctggcgt aatagcgaag 420 aggcccgcac aaaaagggtt ttcccagtca cgacgttgt 45914 458 DNA Artificial Sequence Description of Artificial SequenceArtificially synthesized sequence 14 cgactctaga ggatccccgg gtactttttgttgtgtggaa ttgtgagcgg ataacaattt 60 cacacaggaa acagctatga ccatgattacgaattcgagc tcggtacccg gggatcctct 120 agagtcgacc tgcaggcatg caagcttggcactggccgtc gttttacaac gtcgtgactg 180 ggaaaaccct ggcgttaccc aacttaatcgccttgcagca catccccctt tcgccagctg 240 gcgtaatagc gaagaggccc gcacaaaaagggttttccca gtcacgacgt tgtaaaacga 300 cggccagtgc caagcttgca tgcctgcaggtcgactctag aggatccccg ggtaccgagc 360 tcgaattcgt aatcatggtc atagctgtttcctgtgtgaa attgttatcc gctcacaatt 420 ccacacaaca aaaagtaccc ggggatcctctagagtcg 458 15 790 DNA Artificial Sequence Description of ArtificialSequence Artificially synthesized sequence 15 acaacgtcgt gactgggaaaaccctttttg tgcgggcctc ttcgctatta cgccagctgg 60 cgaaaggggg atgtgctgcaaggcgattaa gttgggtaac gccagggttt tcccagtcac 120 gacgttgtaa aacgacggccagtgccaagc ttgcatgcct gcaggtcgac tctagaggat 180 ccccgggtac cgagctcgaattcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 240 tatccgctca caattccacacaacaaaaag tacccgggga tcctctagag tcgacctgca 300 ggcatgcaag cttggcactggccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 360 ttacccaact taatcgccttgcagcacatc cccctttcgc cagctggcgt aatagcgaag 420 aggcccgcac aaaaagggttttcccagtca cgacgttgta aaacgacggc cagtgccaag 480 cttgcatgcc tgcaggtcgactctagagga tccccgggta ctttttgttg tgtggaattg 540 tgagcggata acaatttcacacaggaaaca gctatgacca tgattacgaa ttcgagctcg 600 gtacccgggg atcctctagagtcgacctgc aggcatgcaa gcttggcact ggccgtcgtt 660 ttacaacgtc gtgactgggaaaaccctggc gttacccaac ttaatcgcct tgcagcacat 720 ccccctttcg ccagctggcgtaatagcgaa gaggcccgca caaaaagggt tttcccagtc 780 acgacgttgt 790 16 789DNA Artificial Sequence Description of Artificial Sequence Artificiallysynthesized sequence 16 cgactctaga ggatccccgg gtactttttg ttgtgtggaattgtgagcgg ataacaattt 60 cacacaggaa acagctatga ccatgattac gaattcgagctcggtacccg gggatcctct 120 agagtcgacc tgcaggcatg caagcttggc actggccgtcgttttacaac gtcgtgactg 180 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagcacatccccctt tcgccagctg 240 gcgtaatagc gaagaggccc gcacaaaaag ggttttcccagtcacgacgt tgtaaaacga 300 cggccagtgc caagcttgca tgcctgcagg tcgactctagaggatccccg ggtaccgagc 360 tcgaattcgt aatcatggtc atagctgttt cctgtgtgaaattgttatcc gctcacaatt 420 ccacacaaca aaaagtaccc ggggatcctc tagagtcgacctgcaggcat gcaagcttgg 480 cactggccgt cgttttacaa cgtcgtgact gggaaaaccctttttgtgcg ggcctcttcg 540 ctattacgcc agctggcgaa agggggatgt gctgcaaggcgattaagttg ggtaacgcca 600 gggttttccc agtcacgacg ttgtaaaacg acggccagtgccaagcttgc atgcctgcag 660 gtcgactcta gaggatcccc gggtaccgag ctcgaattcgtaatcatggt catagctgtt 720 tcctgtgtga aattgttatc cgctcacaat tccacacaacaaaaagtacc cggggatcct 780 ctagagtcg 789 17 310 DNA Artificial SequenceDescription of Artificial Sequence Artificially synthesized sequence 17gtggattcgc actcctcccg ctgatcggga cctgcctcgt cgtctaacaa cagtagtttc 60cggaagtgtt gataagatag gggcatttgg tggtctgtaa gcgggaggag tgcgaatcca 120cactccaaaa gataccaaat actccaaaac agtttctctt ccaaaagtaa ggcaggaaat 180gtgaaaccac aatagttgtc tgatttttag gcccatatta acattgacat agctgactac 240taattccctg gatgctgggt cttccaaatt acgatctggt ttcacatttc ctgccttact 300tttggaagag 310 18 465 DNA Artificial Sequence Description of ArtificialSequence Artificially synthesized sequence 18 gtggattcgc actcctcccgctgatcggga cctgcctcgt cgtctaacaa cagtagtttc 60 cggaagtgtt gataagataggggcatttgg tggtctgtaa gcgggaggag tgcgaatcca 120 cactccaaaa gataccaaatactccaaaac agtttctctt ccaaaagtaa ggcaggaaat 180 gtgaaaccac aatagttgtctgatttttag gcccatatta acattgacat agctgactac 240 taattccctg gatgctgggtcttccaaatt acgatctggt ttcacatttc ctgccttact 300 tttggaagag aaactgttttggagtatttg gtatcttttg gagtgtggat tcgcactcct 360 cccgcttaca gaccaccaaatgcccctatc ttatcaacac ttccggaaac tactgttgtt 420 agacgacgag gcaggtcccgatcagcggga ggagtgcgaa tccac 465 19 20 DNA Bacteriophage M13mp18 19ccggggatcc tctagagtcg 20 20 20 DNA Artificial Sequence Description ofArtificial Sequence Mutant form of M13mp18 20 ccggggatcc tctagagtca 2021 48 DNA Artificial Sequence Description of Artificial SequenceArtificially synthesized primer sequence 21 cgactctaga ggatccccggtttttgttgt gtggaattgt gagcggat 48 22 48 DNA Artificial SequenceDescription of Artificial Sequence Artificially synthesized primersequence 22 tgactctaga ggatccccgg tttttgttgt gtggaattgt gagcggat 48 2347 DNA Artificial Sequence Description of Artificial SequenceArtificially synthesized primer sequence 23 cgtcgtgact gggaaaaccctttttgtgcg ggcctcttcg ctattac 47 24 21 DNA Artificial SequenceDescription of Artificial Sequence Artificially synthesized primersequence 24 actttatgct tccggctcgt a 21 25 17 DNA Artificial SequenceDescription of Artificial Sequence Artificially synthesized primersequence 25 gttgggaagg gcgatcg 17 26 44 DNA Artificial SequenceDescription of Artificial Sequence Artificially synthesized primersequence 26 tgttcctgat gcagtgggca gctttagtct gcggcggtgt tctg 44 27 45DNA Artificial Sequence Description of Artificial Sequence Artificiallysynthesized primer sequence 27 tgctgggtcg gcacagcctg aagctgacctgaaatacctg gcctg 45 28 18 DNA Artificial Sequence Description ofArtificial Sequence Artificially synthesized primer sequence 28tgcttgtggc ctctcgtg 18 29 17 DNA Artificial Sequence Description ofArtificial Sequence Artificially synthesized primer sequence 29gggtgtggga agctgtg 17

1. A method of making a single-stranded nucleic acid molecule havingstem and loop formations at the 5′ and 3′ ends thereof, the methodcomprising: annealing a first oligonucleotide primer to a samplesingle-stranded nucleic acid molecule, the first oligonucleotide primercomprising a 3′ end portion which anneals to the sample single-strandednucleic acid molecule and a 5′ end portion comprising substantially thesame nucleotide sequence as an arbitrary region of the samplesingle-stranded nucleic acid molecule; extending the firstoligonucleotide primer from its 3′ end, using a suitable polymerase, toform a first single-stranded nucleic acid molecule comprising a 5′ endportion comprising a first region and a first complementary regionlocated 5′ terminal which, under suitable conditions, anneal to oneanother to form a loop; displacing the first single-stranded nucleicacid molecule from the sample single-stranded nucleic acid molecule;annealing a second oligonucleotide primer to the first single-strandednucleic acid molecule, the second oligonucleotide primer comprising a 3′end portion which anneals to the first single-stranded nucleic acidmolecule and a 5′ end portion comprising substantially the samenucleotide sequence as an arbitrary region of the first single-strandednucleic acid molecule; extending the second oligonucleotide primer fromits 3′ end, using a suitable polymerase, to form a secondsingle-stranded nucleic acid molecule comprising (i) a 3′ end portioncomplementary to the 5′ end portion of the first single-stranded nucleicacid molecule, the 3′ end portion comprising the first region located 3′terminal and the first complementary region which, under suitablecircumstances, anneal to one another to form a first loop, and (ii) a 5′end portion comprising a second complementary region located 5′ terminaland a second region which, under suitable circumstances, anneal to oneanother to form a second loop; and displacing the second single-strandednucleic acid molecule from the first single-stranded nucleic acidmolecule, whereby the second single-stranded nucleic acid moleculeassumes a conformation with a stem and loop formation formed at both the3′ end portion and the 5′ end portion.
 2. The method according to claim1, wherein each said extending is carried out using a polymerase havingstrand displacement activity.
 3. The method according to claim 1,wherein said displacing the first single-stranded nucleic acid moleculefrom the sample single-stranded nucleic acid molecule comprises:annealing a third oligonucleotide primer to the sample single-strandednucleic acid molecule at a 3′ side position to where the firstoligonucleotide primer anneals thereto; and extending the thirdoligonucleotide primer from its 3′ end, using a polymerase having stranddisplacement activity, to displace the first single-stranded nucleicacid molecule from the sample single-stranded nucleic acid molecule. 4.The method according to claim 1, wherein said displacing the secondsingle-stranded nucleic acid molecule from the first single-strandednucleic acid molecule comprises: annealing a fourth oligonucleotideprimer to the first single-stranded nucleic acid molecule at a 3′ sideposition to where the second oligonucleotide primer anneals thereto; andextending the fourth oligonucleotide primer from its 3′ end, using apolymerase having strand displacement activity, to displace the secondsingle-stranded nucleic acid molecule from the first single-strandednucleic acid molecule.
 5. The method according to claim 1, wherein eachsaid displacing is carried out by heat denaturation.
 6. The methodaccording to claims 1, wherein the sample single-stranded nucleic acidmolecule is RNA, and said extending the first, oligonucleotide primer isconducted by an enzyme having a reverse transcriptase activity.
 7. Themethod according to claim 1 wherein each said extending is carried outin the presence of a melting temperature regulator.
 8. The methodaccording to claim 7, wherein the melting temperature regulator isbetaine.
 9. The method according to claim 8, wherein 0.2 to 3.0 Mbetaine is present.
 10. The method according to claim 1, wherein saiddisplacing the first single-stranded nucleic acid molecule is carriedout beginning from where the 3′ end portion of the first oligonucleotideprimer annealed to the sample single-stranded nucleic acid molecule andcontinuing to the 3′ end of the first single-stranded nucleic acidmolecule.
 11. The method according to claim 1, wherein said displacingthe second single-stranded nucleic acid molecule is carried outbeginning from where the 3′ end portion of the second oligonucleotideprimer annealed to the first single-stranded nucleic acid molecule andcontinuing to the 3′ end of the second single-stranded nucleic acidmolecule.
 12. A method of copying a nucleic acid molecule comprising: A)preparing the second single-stranded nucleic acid molecule according tothe method of claim 1, thereby forming a template; B) extending the 3′terminal of the template to the 5′ end of the template by means of apolymerase having strand displacement activity, when the first regionand first complementary region are annealed to one another to form thefirst loop, to form a template extension which includes the secondcomplementary region and second region located 3′ terminal,respectively, and which under suitable conditions, anneal to one anotherto form a third loop; C) annealing to the first loop of the extendedtemplate an oligonucleotide primer comprising at the 3′ terminal anucleotide sequence complementary to at least part of the first loop andat the 5′ terminal a nucleotide sequence complementary to the firstregion of the template; and D) extending the oligonucleotide primeralong the extended template, by means of a polymerase having stranddisplacement activity, to form a new template complementary to thetemplate formed in step (A).
 13. The method according to claim 12further comprising: E) displacing the new template from the extendedtemplate.
 14. The method according to claim 13 wherein said extending instep (D) displaces the template extension formed during said extendingin step (B), allowing the second complementary region and the secondregion to anneal to one another to form a third loop, said displacing instep (E) comprising: further extending the 3′ terminal of the extendedtemplate to the 5′ end of the template, thereby displacing the newtemplate.
 15. The method according to claim 14 further comprising:annealing to the third loop a second oligonucleotide primer comprisingat the 3′ terminal a nucleotide sequence complementary to at least apart of the third loop and at the 5′ terminal a nucleotide sequencecomplementary to the second region of the template; and extending the 3′terminal of the second oligonucleotide primer by means of a polymerasehaving strand displacement activity.
 16. The method according to claim13, further comprising repeating step (B) through (E) using the newtemplate formed in step (D) as the template.
 17. The method according toclaim 12 wherein each said extending is carried out in the presence of amelting temperature regulator.
 18. The method according to claim 17,wherein the melting temperature regulator is betaine.
 19. The methodaccording to claim 18, wherein 0.2 to 3.0 M betaine is present.
 20. Akit comprising: a first oligonucleotide primer comprising (i) a 3′terminal nucleotide sequence that anneals to a sample single-strandednucleic acid molecule and serves as the origin of synthesis forsynthesizing a first single-stranded nucleic acid molecule complementaryat least in part to the sample single-stranded nucleic acid molecule,and (ii) a 5′ terminal nucleotide sequence that is complementary to anarbitrary region of the first single-stranded nucleic acid molecule; asecond oligonucleotide primer comprising a nucleotide sequence whichanneals to a region of the sample single-stranded nucleic acid moleculelocated 3′ to where the first oligonucleotide primer anneals thereto; athird oligonucleotide primer comprising (i) a 3′ terminal nucleotidesequence that anneals to the first single-stranded nucleic acid moleculeprepared using the first oligonucleotide primer and serves as the originof synthesis for synthesizing a second single-stranded nucleic acidmolecule complementary at least in part to the first single-strandednucleic acid molecule, and (ii) a 5′ terminal nucleotide sequence thatis complementary to an arbitrary region of the second single-strandednucleic acid molecule; a DNA polymerase having strand displacementactivity; and one or more nucleotides which are used by the DNApolymerase to extend the primers.
 21. The kit according to claim 20,further comprising: a fourth oligonucleotide primer comprising anucleotide sequence which anneals to a region of the firstsingle-stranded nucleic acid molecule located 3′ to where the thirdoligonucleotide primer anneals thereto.
 22. The kit according to claim20 further comprising: a detector for detection of a product of nucleicacid synthesis prepared using the remaining components of the kit.
 23. Amethod of making a single-stranded nucleic acid molecule having stem andloop formations at the 5′ and 3′ ends comprising: providing a firstsingle-stranded nucleic acid molecule comprising a 5′ end portioncomprising a first region located 5′ terminal and a first complementaryregion which, under suitable conditions, anneal to one another to form aloop; annealing a first oligonucleotide primer to the firstsingle-stranded nucleic acid molecule, the first oligonucleotide primercomprising a 3′ end portion which anneals to the first single-strandednucleic acid molecule and a 5′ end portion comprising substantially thesame nucleotide sequence as an arbitrary region of the firstsingle-stranded nucleic acid molecule; extending the firstoligonucleotide primer from its 3′ end, using a suitable polymerase, toform a second single-stranded nucleic acid molecule comprising (i) a 3′end portion complementary to the 5′ end portion of the firstsingle-stranded nucleic acid molecule, the 3′ end portion comprising afirst region located 3′ terminal and a first complementary region which,under suitable circumstances, anneal to one another to form a firstloop, and (ii) a 5′ end portion comprising a second region located 5′terminal and a second complementary region which, under suitablecircumstances, anneal to one another to form a second loop; displacingthe second single-stranded nucleic acid molecule from the firstsingle-stranded nucleic acid molecule, beginning from where the 3′ endportion of the first oligonucleotide primer annealed to the firstsingle-stranded nucleic acid molecule and continuing to the 3′ end ofthe second nucleic acid molecule, wherein, upon displacement of thesecond single-stranded nucleic acid molecule from the firstsingle-stranded nucleic acid molecule, the second single-strandednucleic acid molecule assumes a conformation having the 3′ end portionforming the first loop and the 5′ end portion forming the second loop.24. The method according to claim 23 wherein said extending is carriedout using a polymerase having strand displacement activity.
 25. Themethod according to claim 23 wherein said annealing and said extendingare carried out in the presence of a melting temperature regulator. 26.The method according to claim 25 wherein the melting temperatureregulator is betaine.
 27. The method according to claim 26, wherein 0.2to 3.0 M betaine is present.
 28. A method of copying a nucleic acidcomprising: A) preparing the second single-stranded nucleic acidmolecule according to the method of claim 23, thereby forming atemplate; B) extending the 3′ terminal of the template to the 5′ end ofthe template by means of a polymerase having strand displacementactivity, when the first region and first complementary region areannealed to one another to form the first loop, to form a templateextension which includes a third region located 3′ terminal and a thirdcomplementary region which are substantially the same as the secondcomplementary region and second region, respectively, and which, undersuitable conditions, anneal to one another to form a third loop; C)annealing to the first loop of the extended template an oligonucleotideprimer comprising at the 3′ terminal a nucleotide sequence complementaryto at least part of the first loop and at the 5′ terminal a nucleotidesequence complementary to the first region of the template; and D)extending the oligonucleotide primer along the extended template, bymeans of a polymerase having strand displacement activity, to form a newtemplate complementary to the template formed in step (A).
 29. Themethod according to claim 28 further comprising: E) displacing the newtemplate from the extended template.
 30. The method according to claim29 wherein said extending in step (D) displaces the template extensionformed during said extending in step (B), allowing the third region andthe third complementary region to anneal to one another to form a thirdloop, said displacing in step (E) comprising: further extending the 3′terminal of the extended template to the 5′ end of the template, therebydisplacing the new template.
 31. The method according to claim 30further comprising: annealing to the third loop a second oligonucleotideprimer comprising at the 3′ terminal a nucleotide sequence complementaryto at least a part of the third loop and at the 5′ terminal a nucleotidesequence complementary to the third region of the template; andextending the 3′ terminal of the second oligonucleotide primer by meansof a polymerase having strand displacement activity.
 32. The methodaccording to claim 29, wherein the new template has (i) a 5′ end portioncomprising the first region and the first complementary region located5′ terminal which, under suitable conditions, anneal to one another toform the first loop, and (ii) a 3′ end portion comprising the secondregion and the second complementary region located 3′ terminal which,under suitable conditions, anneal to one another to form the secondloop, said method further comprising: F) extending the 3′ terminal ofthe new template to the 5′ end of the new template by means of apolymerase having strand displacement activity, when the second regionand second complementary region are annealed to one another to form thesecond loop, to form a template extension which includes a third regionand a third complementary region that are substantially the same as thefirst complementary region and first region, respectively, and which,under suitable conditions, anneal to one another to form a third loop;G) annealing to the second loop of the extended new template a secondoligonucleotide primer comprising at the 3′ terminal a nucleotidesequence complementary to at least a part of the second loop and at the5′ terminal a nucleotide sequence complementary to the secondcomplementary region of the template; H) extending the secondoligonucleotide primer along the extended new template, by means of apolymerase having strand displacement activity, to form a third templatewhich is substantially the same as the template.
 33. The methodaccording to claim 32 further comprising: I) displacing the thirdtemplate from the new template.
 34. The method according to claim 33further comprising: repeating steps (B) through (I) using the thirdtemplate.
 35. The method according to claim 28 wherein each saidextending is carried out in the presence of a melting temperatureregulator.
 36. The method according to claim 35, wherein the meltingtemperature regulator is betaine.
 37. The method according to claim 36,wherein 0.2 to 3.0 M betaine is present.
 38. A method of making asingle-stranded nucleic acid molecule having stem and loop formations atthe 5′ and 3′ ends thereof, the method comprising: annealing a firstoligonucleotide primer to a sample single-stranded nucleic acidmolecule, the first oligonucleotide primer comprising a 3′ end portionwhich anneals to the sample single-stranded nucleic acid molecule and a5′ end portion comprising substantially the same nucleotide sequence asan arbitrary region of the sample single-stranded nucleic acid molecule;extending the first oligonucleotide primer from its 3′ end, using asuitable polymerase, to form a first single-stranded nucleic acidmolecule comprising a 5′ end portion comprising a first region and afirst complementary region located 5′ terminal which, under suitableconditions, anneal to one another to form a loop; displacing the firstsingle-stranded nucleic acid molecule from the sample single-strandednucleic acid molecule, beginning from where the 3′ end portion of thefirst oligonucleotide primer annealed to the sample single-strandednucleic acid molecule and continuing to the 3′ end of the firstsingle-stranded nucleic acid molecule; annealing a secondoligonucleotide primer to the first single-stranded nucleic acidmolecule, the second oligonucleotide primer comprising a 3′ end portionwhich anneals to the first single-stranded nucleic acid molecule and a5′ end portion comprising substantially the same nucleotide sequence asan arbitrary region of the first single-stranded nucleic acid molecule;extending the second oligonucleotide primer from its 3′ end, using asuitable polymerase, to form a second single-stranded nucleic acidmolecule comprising (i) a 3′ end portion complementary to the 5′ endportion of the first single-stranded nucleic acid molecule, the 3′ endportion comprising the first region located 3′ terminal and the firstcomplementary region which, under suitable circumstances, anneal to oneanother to form a first loop, and (ii) a 5′ end portion comprising asecond region located 5′ terminal and a second complementary regionwhich, under suitable circumstances, anneal to one another to form asecond loop; and displacing the second single-stranded nucleic acidmolecule from the first single-stranded nucleic acid molecule, wherebythe second single-stranded nucleic acid molecule assumes a conformationwith a stem and loop formation formed at both the 3′ end portion and the5′ end portion.
 39. The method according to claim 38, wherein each saidextending is carried out using a polymerase having strand displacementactivity.
 40. The method according to claim 38, wherein each saidannealing and each said extending is carried out in the presence of amelting temperature regulator.
 41. The method according to claim 40,wherein the melting temperature regulator is betaine.
 42. The methodaccording to claim 41, wherein 0.2 to 3.0 M betaine is present.
 43. Themethod according to claim 38, wherein said displacing the secondsingle-stranded nucleic acid molecule is carried out beginning fromwhere the 3′ end portion of the second oligonucleotide primer annealedto the first single-stranded nucleic acid molecule and continuing to the3′ end of the second single-stranded nucleic acid molecule.
 44. A methodof copying a nucleic acid molecule comprising: A) preparing the secondsingle-stranded nucleic acid molecule according to the method of claim38, thereby forming a template; B) extending the 3′ terminal of thetemplate to the 5′ end of the template by means of a polymerase havingstrand displacement activity, when the first region and firstcomplementary region are annealed to one another to form the first loop,to form a template extension which includes a third region located 3′terminal and a third complementary region which are substantially thesame as the second complementary region and second region, respectively,and which, under suitable conditions, anneal to one another to form athird loop; C) annealing to the first loop of the extended template anoligonucleotide primer comprising at the 3′ terminal a nucleotidesequence complementary to at least part of the first loop and at the 5′terminal a nucleotide sequence complementary to the first region of thetemplate; and D) extending the oligonucleotide primer along the extendedtemplate, by means of a polymerase having strand displacement activity,to form a new template complementary to the template formed in step (A).45. The method according to claim 44 further comprising: E) displacingthe new template from the extended template.
 46. The method according toclaim 45 wherein said extending in step (D) displaces the templateextension formed during said extending in step (B), allowing the thirdregion and the third complementary region to anneal to one another toform a third loop, said displacing in step (E) comprising: furtherextending the 3′ terminal of the extended template to the 5′ end of thetemplate, thereby displacing the new template.
 47. The method accordingto claim 46 further comprising: annealing to the third loop a secondoligonucleotide primer comprising at the 3′ terminal a nucleotidesequence complementary to at least a part of the third loop and at the5′ terminal a nucleotide sequence complementary to the third region ofthe template; and extending the 3′ terminal of the secondoligonucleotide primer by means of a polymerase having stranddisplacement activity.
 48. The method according to claim 44, wherein thenew template has (i) a 5′ end portion comprising the first region andthe first complementary region located 5′ terminal which, under suitableconditions, anneal to one another to form the first loop, and (ii) a 3′end portion comprising the second region and the second complementaryregion located 3′ terminal which, under suitable conditions, anneal toone another to form the second loop, said method further comprising: F)extending the 3′ terminal of the new template to the 5′ end of the newtemplate by means of a polymerase having strand displacement activity,when the second region and second complementary region are annealed toone another to form the second loop, to form a template extension whichincludes a third region and a third complementary region that aresubstantially the same as the first complementary region and firstregion, respectively, and which, under suitable conditions, anneal toone another to form a third loop; G) annealing to the second loop of theextended new template a second oligonucleotide primer comprising at the3′ terminal a nucleotide sequence complementary to at least a part ofthe second loop and at the 5′ terminal a nucleotide sequencecomplementary to the second complementary region of the template; H)extending the second oligonucleotide primer along the extended newtemplate, by means of a polymerase having strand displacement activity,to form a third template which is substantially the same as thetemplate.
 49. The method according to claim 48 further comprising: I)displacing the third template from the new template.
 50. The methodaccording to claim 49 further comprising: repeating steps (B) through(I) using the third template.
 51. The method according to claim 44wherein each said extending is carried out in the presence of a meltingtemperature regulator.
 52. The method according to claim 51, wherein themelting temperature regulator is betaine.
 53. The method according toclaim 52, wherein 0.2 to 3.0 M betaine is present.